Protein kinase homologs

ABSTRACT

The invention provides human protein kinase homologs (PKH) and polynucleotides which identify and encode PKH. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of PKH.

This application is a divisional application of U.S. application Ser.No. 09/173,581, filed Oct. 15, 1998 now U.S. Pat. No. 6,013,455 issuedJan. 11, 2000.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences ofprotein kinase homologs and to the use of these sequences in thediagnosis, treatment, and prevention of cancer, autoimmune/inflammatorydisorders, and reproductive disorders.

BACKGROUND OF THE INVENTION

Kinases and phosphatases are critical components of intracellular signaltransduction mechanisms. Kinases catalyze the transfer of high energyphosphate groups from adenosine triphosphate (ATP) to hydroxyamino acidsof various target proteins. Phosphatases, in contrast, remove phosphategroups from proteins. Reversible protein phosphorylation is the mainstrategy for regulating protein activity in eukaryotic cells. Ingeneral, proteins are activated by phosphorylation in response toextracellular signals such as hormones, neurotransmitters, and growthand differentiation factors. Protein dephosphorylation occurs whendown-regulation of a signaling pathway is required. The combinedactivities of kinases and phosphatases regulate key cellular processessuch as proliferation, differentiation, and cell cycle progression.

Kinases comprise the largest known enzyme superfamily and vary widely intheir target profeins. Kinases may be categorized as protein tyrosinekinases (PTKs), which phosphorylate tyrosine residues, and proteinserine/threonine kinases (STKs), which phosphorylate serine and/orthreonine residues. Some kinases have dual specificity for bothserine/threonine and tyrosine residues. Almost all kinases contain aconserved 250-300 amino acid catalytic domain. This domain can befurther divided into 11 subdomains. N-terminal subdomains I-IV fold intoa two-lobed structure which binds and orients the ATP donor molecule,and subdomain V spans the two lobes. C-terminal subdomains VI-XI bindthe protein substrate and transfer the gamma phosphate from ATP to thehydroxyl group of a serine, threonine, or tyrosine residue. Each of the11 subdomains contains specific catalytic residues or amino acid motifscharacteristic of that subdomain. For example, subdomain I contains an8-amino acid glycine-rich ATP binding consensus motif, subdomain IIcontains a critical lysine residue required for maximal catalyticactivity, and subdomains VI through IX comprise the highly conservedcatalytic core. STKs and PTKs also contain distinct sequence motifs insubdomains VI and VIII which may confer hydroxyamino acid specificity.Some STKs and PTKs possess structural characteristics of both families.In addition, kinases may also be classified by additional amino acidsequences, generally between 5 and 100 residues, which either flank oroccur within the kinase domain. These additional amino acid sequencesregulate kinase activity and determine substrate specificity. (Reviewedin Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book, VolI:7-20 Academic Press, San Diego, Calif.)

PTKs may be classified as either transmembrane or non-transmembraneproteins. Transmembrane tyrosine kinases function as receptors for mostgrowth factors. Binding of growth factor to the receptor activates thetransfer of a phosphate group from ATP to selected tyrosine residues inthe receptor itself and in specific second messenger proteins. Growthfactors (GF) that associate with receptor PTKs include epidermal GF,platelet-derived GF, fibroblast GF, hepatocyte GF, insulin andinsulin-like GFs, nerve GF, vascular endothelial GF, and macrophagecolony stimulating factor.

Non-transmembrane PTKs form signaling complexes with the cytosolicdomains of plasma membrane receptors. Receptors that signal throughnon-transmembrane PTKs include cytokine, hormone, and antigen-specificlymphocytic receptors. Many PTKs were first identified as oncogeneproducts in cancer cells in which PTK activation was no longer subjectto normal cellular controls. In fact, about one third of the knownoncogenes encode PTKs. Furthermore, cellular transformation(oncogenesis) is often accompanied by increased tyrosine phosphorylationactivity. (Carbonneau, H. and Tonks, N. K. (1992) Annu. Rev. Cell Biol.8:463-93.) Regulation of PTK activity may therefore be an importantstrategy in controlling some types of cancer.

The discovery of new protein kinase homologs and the polynucleotidesencoding them satisfies a need in the art by providing new compositionswhich are useful in the diagnosis, prevention, and treatment of cancer,autoimmune/inflammatory disorders, and reproductive disorders.

SUMMARY OF THE INVENTION

The invention features substantially purified polypeptides, proteinkinase homologs, referred to collectively as “PKH” and individually as“PKH-1”, “PKH-2”, “PKH-3”, “PKH-4”, “PKH-5”, “PKH-6”, “PKH-7”, “PKH-8”,and “PKH-9”. In one aspect, the invention provides a substantiallypurified polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, (SEQ IDNO: 1-9), and fragments thereof.

The invention further provides a substantially purified variant havingat least 90% amino acid identity to at least one of the amino acidsequences selected from the group consisting of SEQ ID NO: 1-9, andfragments thereof. The invention also provides an isolated and purifiedpolynucleotide encoding the polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 1-9, andfragments thereof. The invention also includes an isolated and purifiedpolynucleotide variant having at least 70% polynucleotide sequenceidentity to the polynucleotide encoding the polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1-9, and fragments thereof.

Additionally, the invention provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 1-9, andfragments thereof. The invention also provides an isolated and purifiedpolynucleotide having a sequence which is complementary to thepolynucleotide encoding the polypeptide comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 1-9, andfragments thereof.

The invention also provides an isolated and purified polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, (SEQID NO: 10-18) and fragments thereof. The invention further provides anisolated and purified polynucleotide variant having at least 70%polynucleotide sequence identity to the polynucleotide sequence selectedfrom the group consisting of SEQ ID NO: 10-18, and fragments thereof.The invention also provides an isolated and purified polynucleotidehaving a sequence which is complementary to the polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO: 10-18, and fragments thereof.

The invention also provides a method for detecting a polynucleotide in asample containing nucleic acids, the method comprising the steps of (a)hybridizing the complement of the polynucleotide sequence to at leastone of the polynucleotides of the sample, thereby forming ahybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide in the sample. In one aspect, the methodfurther comprises amplifying the polynucleotide prior to hybridization.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1-9, and fragments thereof. In another aspect, the expression vector iscontained within a host cell.

The invention also provides a method for producing a polypeptide, themethod comprising the steps of: (a) culturing the host cell containingan expression vector containing at least a fragment of a polynucleotideunder conditions suitable for the expression of the polypeptide; and (b)recovering the polypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequenceselected from the group consisting of SEQ ID NO: 1-9, and fragmentsthereof, in conjunction with a suitable pharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide selected from the group consisting of SEQ ID NO: 1-9, andfragments thereof. The invention also provides a purified agonist and apurified antagonist to the polypeptide.

The invention also provides a method for treating or preventing adisorder of cell proliferation associated with decreased expression oractivity of PKH, the method comprising administering to a subject inneed of such treatment an effective amount of a pharmaceuticalcomposition comprising a substantially purified polypeptide having theamino acid sequence selected from the group consisting of SEQ ID NO:1-9, and fragments thereof, in conjunction with a suitablepharmaceutical carrier.

The invention also provides a method for treating or preventing adisorder of cell proliferation associated with increased expression oractivity of PKH, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-9, and fragments thereof.

BRIEF DESCRIPTION OF THE TABLES

Table 1 shows nucleotide and polypeptide sequence identification numbers(SEQ ID NO), clone identification numbers (clone ID), cDNA libraries,and cDNA fragments used to assemble full-length sequences encoding PKH.

Table 2 shows features of each polypeptide sequence including potentialmotifs, homologous sequences, and methods and algorithms used forcharacterization of PKH.

Table 3 shows the tissue-specific expression patterns of each nucleicacid sequence as determined by northern analysis, diseases, disorders,or conditions associated with these tissues, and the vector into whicheach cDNA was cloned.

Table 4 describes the tissues used to construct the cDNA libraries fromwhich Incyte cDNA clones encoding PKH were isolated.

Table 5 shows the programs, their descriptions, references, andthreshold parameters used to analyze PKH.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular machines, materials and methods described, as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

DEFINITIONS

“PKH” refers to the amino acid sequences of substantially purified PKHobtained from any species, particularly a mammalian species, includingbovine, ovine, porcine, murine, equine, and preferably the humanspecies, from any source, whether natural, synthetic, semi-synthetic, orrecombinant.

The term “agonist” refers to a molecule which, when bound to PKH,increases or prolongs the duration of the effect of PKH. Agonists mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to and modulate the effect of PKH.

An “allelic variant” is an alternative form of the gene encoding PKH.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. Any given natural orrecombinant gene may have none, one, or many allelic forms. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding PKH include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polynucleotide the same as PKH or a polypeptide with atleast one functional characteristic of PKH. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingPKH, and improper or unexpected hybridization to allelic variants, witha locus other than the normal chromosomal locus for the polynucleotidesequence encoding PKH. The encoded protein may also be “altered,” andmay contain deletions, insertions, or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent PKH. Deliberate amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological or immunological activity of PKH is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid, positively charged amino acids may include lysine andarginine, and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules. Inthis context, “fragments,” “immunogenic fragments,” or “antigenicfragments” refer to fragments of PKH which are preferably at least 5 toabout 15 amino acids in length, most preferably at least 14 amino acids,and which retain some biological activity or immunological activity ofPKH. Where “amino acid sequence” is recited to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms are not meant to limit the amino acid sequenceto the complete native amino acid sequence associated with the recitedprotein molecule.

“Amplification” relates to the production of additional copies of anucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

The term “antagonist” refers to a molecule which, when bound to PKH,decreases the amount or the duration of the effect of the biological orimmunological activity of PKH. Antagonists may include proteins, nucleicacids, carbohydrates, antibodies, or any other molecules which decreasethe effect of PKH.

The term “antibody” refers to intact molecules as well as to fragmentsthereof, such as Fab, F(ab′)₂, and Fv fragments, which are capable ofbinding the epitopic determinant. Antibodies that bind PKH polypeptidescan be prepared using intact polypeptides or using fragments containingsmall peptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal (e.g., a mouse, a rat, or arabbit) can be derived from the translation of RNA, or synthesizedchemically, and can be conjugated to a carrier protein if desired.Commonly used carriers that are chemically coupled to peptides includebovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin(KLH). The coupled peptide is then used to immunize the animal.

The term “antigenic determinant” refers to that fragment of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (given regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “antisense” refers to any composition containing a nucleic acidsequence which is complementary to the “sense” strand of a specificnucleic acid sequence. Antisense molecules may be produced by any methodincluding synthesis or transcription. Once introduced into a cell, thecomplementary nucleotides combine with natural sequences produced by thecell to form duplexes and to block either transcription or translation.The designation “negative” can refer to the antisense strand, and thedesignation “positive” can refer to the sense strand.

The term “biologically active,” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” refers to the capability of thenatural, recombinant, or synthetic PKH, or of any oligopeptide thereof,to induce a specific immune response in appropriate animals or cells andto bind with specific antibodies.

The terms “complementary” or “complementarity” refer to the naturalbinding of polynucleotides by base pairing. For example, the sequence“5′ A-G-T 3′” bonds to the complementary sequence “3′ T-C-A 5′.”Complementarity between two single-stranded molecules may be “partial,”such that only some of the nucleic acids bind, or it may be “complete,”such that total complementarity exists between the single strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encoding PKHor fragments of PKH may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beenresequenced to resolve uncalled bases, extended using XL-PCR kit(Perkin-Elmer, Norwalk Conn.) in the 5′ and/or the 3′ direction, andresequenced, or which has been assembled from the overlapping sequencesof more than one Incyte Clone using a computer program for fragmentassembly, such as the GELVIEW fragment assembly system (GCG, MadisonWis.). Some sequences have been both extended and assembled to producethe consensus sequence.

The term “correlates with expression of a polynucleotide” indicates thatthe detection of the presence of nucleic acids, the same or related to anucleic acid sequence encoding PKH, by northern analysis is indicativeof the presence of nucleic acids encoding PKH in a sample, and therebycorrelates with expression of the transcript from the polynucleotideencoding PKH.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to the chemical modification of apolypeptide sequence, or a polynucleotide sequence. Chemicalmodifications of a polynucleotide sequence can include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A derivativepolynucleotide encodes a polypeptide which retains at least onebiological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

The term “similarity” refers to a degree of complementarity. There maybe partial similarity or complete similarity. The word “identity” maysubstitute for the word “similarity.” A partially complementary sequencethat at least partially inhibits an identical sequence from hybridizingto a target nucleic acid is referred to as “substantially similar.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or northern blot, solution hybridization, and the like) underconditions of reduced stringency. A substantially similar sequence orhybridization probe will compete for and inhibit the binding of acompletely similar (identical) sequence to the target sequence underconditions of reduced stringency. This is not to say that conditions ofreduced stringency are such that non-specific binding is permitted, asreduced stringency conditions require that the binding of two sequencesto one another be a specific (i.e., a selective) interaction. Theabsence of non-specific binding may be tested by the use of a secondtarget sequence which lacks even a partial degree of complementarity(e.g., less than about 30% similarity or identity). In the absence ofnon-specific binding, the substantially similar sequence or probe willnot hybridize to the second non-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR, MadisonWis.) which creates alignments between two or more sequences accordingto methods selected by the user, e.g., the clustal method. (See, e.g.,Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustalalgorithm groups sequences into clusters by examining the distancesbetween all pairs. The clusters are aligned pairwise and then in groups.The percentage similarity between two amino acid sequences, e.g.,sequence A and sequence B, is calculated by dividing the length ofsequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no similarity between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweennucleic acid sequences can also be counted or calculated by othermethods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein,J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences canalso be determined by other methods known in the art, e.g., by varyinghybridization conditions.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size, and whichcontain all of the elements required for stable mitotic chromosomesegregation and maintenance.

The term “humanized antibody” refers to antibody molecules in which theamino acid sequence in the non-antigen binding regions has been alteredso that the antibody more closely resembles a human antibody, and stillretains its original binding ability.

“Hybridization” refers to any process by which a strand of nucleic acidbinds with a complementary strand through base pairing.

The term “hybridization complex” refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary bases. A hybridization complex may be formed insolution (e.g., C₀t or R₀t analysis) or formed between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., paper, membranes, filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed).

The words “insertion” or “addition” refer to changes in an amino acid ornucleotide sequence resulting in the addition of one or more amino acidresidues or nucleotides, respectively, to the sequence found in thenaturally occurring molecule.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term “microarray” refers to an arrangement of distinctpolynucleotides on a substrate.

The terms “element” or “array element” in a microarray context, refer tohybridizable polynucleotides arranged on the surface of a substrate.

The term “modulate” refers to a change in the activity of PKH. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of PKH.

The phrases “nucleic acid” or “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material. In this context, “fragments” refers tothose nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

The terms “operably associated” or “operably linked” refer tofunctionally related nucleic acid sequences. A promoter is operablyassociated or operably linked with a coding sequence if the promotercontrols the translation of the encoded polypeptide. While operablyassociated or operably linked nucleic acid sequences can be contiguousand in the same reading frame, certain genetic elements, e.g., repressorgenes, are not contiguously linked to the sequence encoding thepolypeptide but still bind to operator sequences that control expressionof the polypeptide.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to 60 nucleotides, preferably about 15 to 30nucleotides, and most preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or in a hybridization assay or microarray.“Oligonucleotide” is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining nucleic acids encoding PKH, or fragments thereof, or PKHitself, may comprise a bodily fluid; an extract from a cell, chromosome,organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA,or cDNA, in solution or bound to a substrate; a tissue; a tissue print;etc.

The terms “specific binding” or “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, oran antagonist. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide containingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “stringent conditions” refers to conditions which permithybridization between polynucleotides and the claimed polynucleotides.Stringent conditions can be defined by salt concentration, theconcentration of organic solvent, e.g., formamide, temperature, andother conditions well known in the art. In particular, stringency can beincreased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably about75% free, and most preferably about 90% free from other components withwhich they are naturally associated.

A “substitution” refers to the replacement of one or more amino acids ornucleotides by different amino acids or nucleotides, respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

“Transformation” describes a process by which exogenous DNA enters andchanges a recipient cell. Transformation may occur under natural orartificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, viralinfection, electroporation, heat shock, lipofection, and particlebombardment. The term “transformed” cells includes stably transformedcells in which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome, aswell as transiently transformed cells which express the inserted DNA orRNA for limited periods of time.

A “variant” of PKH polypeptides refers to an amino acid sequence that isaltered by one or more amino acid residues. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software (DNASTAR).

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to PKH. Thisdefinition may also include, for example, “allelic” (as defined above),“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. The resulting polypeptides generally will have significantamino acid identity relative to each other. A polymorphic variant is avariation in the polynucleotide sequence of a particular gene betweenindividuals of a given species. Polymorphic variants also may encompass“single nucleotide polymorphisms” (SNPS) in which the polynucleotidesequence varies by one base. The presence of SNPs may be indicative of,for example, a certain population, a disease state, or a propensity fora disease state.

THE INVENTION

The invention is based on the discovery of new human protein kinasehomologs (PKH), the polynucleotides encoding PKH, and the use of thesecompositions for the diagnosis, treatment, or prevention of cancer,autoimmune/inflammatory disorders, and reproductive disorders.

Table 1 lists the Incyte Clones used to derive full length nucleotidesequences encoding PKH. Columns 1 and 2 show the sequence identificationnumbers (SEQ ID NO) of the amino acid and nucleic acid sequences,respectively. Column 3 shows the Clone ID of the Incyte Clone in whichnucleic acids encoding each PKH were identified, and column 4, the cDNAlibraries from which these clones were isolated. Column 5 shows Incyteclones, their corresponding cDNA libraries, and shotgun sequences. Theclones and shotgun sequences are part of the consensus nucleotidesequence of each PKH and are useful as fragments in hybridizationtechnologies.

The columns of Table 2 show various properties of the polypeptides ofthe invention: column 1 references the amino acid SEQ ID NO; column 2shows the number of amino acid residues in each polypeptide; column 3,potential phosphorylation sites; column 4, the amino acid residuescomprising signature sequences and motifs; column 5, the identity ofeach protein; and column 6, analytical methods used to characterize andidentify each protein through sequence homology and protein motifs.

The columns of Table 3 show the tissue specificity and diseases,disorders, or conditions associated with nucleotide sequences encodingPKH. The first column of Table 3 lists the nucleotide SEQ ID NO; thesecond column lists tissue categories which express PKH as a fraction oftotal tissue categories expressing PKH. The third column lists thediseases, disorders, or conditions associated with those tissuesexpressing PKH. The fourth column lists the vectors used to subclone thecDNA library.

The following fragments of the nucleotide sequences encoding PKH areuseful in hybridization or amplification technologies to identify SEQ IDNO: 10-18 and to distinguish between SEQ ID NO: 10-18 and relatedpolynucleotide sequences. The useful fragments are the fragment of SEQID NO: 10 from about nucleotide 473 to about nucleotide 532; thefragment of SEQ ID NO: 11 from about nucleotide 65 to about nucleotide125; the fragment of SEQ ID NO:12 from about nucleotide 96 to aboutnucleotide 155; the fragment of SEQ ID NO:13 from about nucleotide 805to about nucleotide 864; the fragment of SEQ ID NO: 14 from aboutnucleotide 230 to about nucleotide 289; the fragment of SEQ ID NO: 15from about nucleotide 154 to about nucleotide 213; the fragment of SEQID NO:16 from about nucleotide 110 to about nucleotide 169; the fragmentof SEQ ID NO: 17 from about nucleotide 482 to about nucleotide 541; andthe fragment of SEQ ID NO: 18 from about nucleotide 115 to aboutnucleotide 174.

The invention also encompasses PKH variants. A preferred PKH variant isone which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe PKH amino acid sequence, and which contains at least one functionalor structural characteristic of PKH.

The invention also encompasses polynucleotides which encode PKH. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO: 10-18, which encodes PKH.

The invention also encompasses a variant of a polynucleotide sequenceencoding PKH. In particular, such a variant polynucleotide sequence willhave at least about 70%, more preferably at least about 85%, and mostpreferably at least about 95% polynucleotide sequence identity to thepolynucleotide sequence encoding PKH. A particular aspect of theinvention encompasses a variant of a polynucleotide sequence comprisinga sequence selected from the group consisting of SEQ ID NO: 10-18 whichhas at least about 70%, more preferably at least about 85%, and mostpreferably at least about 95% polynucleotide sequence identity to anucleic acid sequence selected from the group consisting of SEQ ID NO:10-18. Any one of the polynucleotide variants described above can encodean amino acid sequence which contains at least one functional orstructural characteristic of PKH.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding PKH, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring PKH, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode PKH and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring PKH under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding PKH or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding PKH and its derivatives without altering the encoded amino acidsequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodePKH and PKH derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding PKH or any fragmentthereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO: 10-18 and fragments thereofunder various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.) For example, stringent salt concentrationwill ordinarily be less than about 750 mM NaCl and 75 mM trisodiumcitrate, preferably less than about 500 mM NaCl and 50 mM trisodiumcitrate, and most preferably less than about 250 mM NaCl and 25 mMtrisodium citrate. Low stringency hybridization can be obtained in theabsence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and most preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μ/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Perkin-Elmer),thermostable T7 polymerase (Amersham Pharmacia Biotech, PiscatawayN.J.), or combinations of polymerases and proofreading exonucleases suchas those found in the ELONGASE amplification system (Life Technologies,Gaithersburg Md.). Preferably, sequence preparation is automated withmachines such as the MICROLAB 2200 (Hamilton, Reno Nev.), Peltierthermal cycler 200 (PTC200; MJ Research, Watertown Mass.) and the ABICATALYST 800 (Perkin-Elmer). Sequencing is then carried out using eitherABI 373 or 377 DNA sequencing systems (Perkin-Elmer) or the MEGABACE1000 DNA sequencing system (Molecular Dynamics, Sunnyvale Calif.). Theresulting sequences are analyzed using a variety of algorithms which arewell known in the art. (See, e.g., Ausubel, F. M. (1997) Short Protocolsin Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7;Meyers, R. A. (1995) Molecular Biolog and Biotechnology, Wiley VCH, NewYork N.Y., pp. 856-853.)

The nucleic acid sequences encoding PKH may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 Primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR,Perkin-Elmer), and the entire process from loading of samples tocomputer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode PKH may be cloned in recombinant DNAmolecules that direct expression of PKH, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express PKH.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter PKH-encodingsequences for a variety of purposes including, but not limited to,modification of the cloning, processing, and/or expression of the geneproduct. DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides may be used to engineerthe nucleotide sequences. For example, oligonucleotide-mediatedsite-directed mutagenesis may be used to introduce mutations that createnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

In another embodiment, sequences encoding PKH may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, PKH itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A Peptide synthesizer (Perkin-Elmer).Additionally, the amino acid sequence of PKH, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, WH Freeman, New York N.Y.)

In order to express a biologically active PKH, the nucleotide sequencesencoding PKH or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesequences encoding PKH. Such elements may vary in their strength andspecificity. Specific initiation signals may also be used to achievemore efficient translation of sequences encoding PKH. Such signalsinclude the ATG initiation codon and adjacent sequences, e.g. the Kozaksequence. In cases where sequences encoding PKH and its initiation codonand upstream regulatory sequences are inserted into the appropriateexpression vector, no additional transcriptional or translationalcontrol signals may be needed. However, in cases where only codingsequence, or a fragment thereof, is inserted, exogenous translationalcontrol signals including an in-frame ATG initiation codon should beprovided by the vector. Exogenous translational elements and initiationcodons may be of various origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of enhancersappropriate for the particular host cell system used. (See, e.g.,Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding PKH andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, old Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding PKH. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding PKH. For example, routine cloning, subcloning, and propagationof polynucleotide sequences encoding PKH can be achieved using amultifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JollaCalif.) or PSPORT1 plasmid (Life Technologies). Ligation of sequencesencoding PKH into the vector's multiple cloning site disrupts the lacZgene, allowing a colorimetric screening procedure for identification oftransformed bacteria containing recombinant molecules. In addition,these vectors may be useful for in vitro transcription, dideoxysequencing, single strand rescue with helper phage, and creation ofnested deletions in the cloned sequence. (See, e.g., Van Heeke, G. andS. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of PKH are needed, e.g. for the production of antibodies,vectors which direct high level expression of PKH may be used. Forexample, vectors containing the strong, inducible T5 or T7 bacteriophagepromoter may be used.

Yeast expression systems may be used for production of PKH. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH, may be used in the yeast Saccharomycescerevisiae or Pichia Rastoris. In addition, such vectors direct eitherthe secretion or intracellular retention of expressed proteins andenable integration of foreign sequences into the host genome for stablepropagation. (See, e.g., Ausubel, 1995, supra; Grant et al. (1987)Methods Enzymol. 153:516-54; and Scorer, C. A. et al. (1994)Bio/Technology 12:181-184.)

Plant systems may also be used for expression of PKH. Transcription ofsequences encoding PKH may be driven viral promoters, e.g., the 35S and19S promoters of CaMV used alone or in combination with the omega leadersequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., The McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New YorkN.Y., pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding PKH may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses PKH in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 8-1:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. etal. (1997) Nat Genet. 15:345-355.)

For long term production of recombinant proteins in mammalian systems,stable expression of PKH in cell lines is preferred. For example,sequences encoding PKH can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ or apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the. aminoglycosides, neomycinand G-418; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F.et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988)Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,anthocyanins, green fluorescent proteins (GFP; Clontech), βglucuronidase and its substrate β-glucuronide, or luciferase and itssubstrate luciferin may be used. These markers can be used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system.(See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encoding PKHis inserted within a marker gene sequence, transformed cells containingsequences encoding PKH can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding PKH under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingPKH and that express PKH may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification,and protein bioassay or immunoassay techniques which include membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of PKHusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-linked immunosorbentassays (ELISAs), radioimmunoassays (RIAs), and fluorescence activatedcell sorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on PKH ispreferred, but a competitive binding assay may be employed. These andother assays are well known in the art. (See, e.g., Hampton, R. et al.(1990) Serological Methods, a Laboratory Manual, APS Press, St PaulMinn., Sect. IV; Coligan, J. E. et al. (1997) Current Protocols inImmunology, Greene Pub. Associates and Wiley-Interscience, New YorkN.Y.; and Pound, J. D. (1998) Immunochemical Protocols, Humana Press,Totowa N.J.).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding PKH includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding PKH,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamPharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitablereporter molecules or labels which may be used for ease of detectioninclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding PKH may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodePKH may be designed to contain signal sequences which direct secretionof PKH through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC, Manassas Va.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding PKH may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric PKHprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of PKH activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the PKH encodingsequence and the heterologous protein sequence, so that PKH may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel (1995, supra, ch 10). A variety of commercially available kitsmay also be used to facilitate expression and purification of fusionproteins.

In a further embodiment of the invention, synthesis of radiolabeled PKHmay be achieved in vitro using the TNT rabbit reticulocyte lysate orwheat germn extract systems (Promega). These systems coupletranscription and translation of protein-coding sequences operablyassociated with the T7, T3, or SP6 promoters. Translation takes place inthe presence of a radiolabeled amino acid precursor, preferably³⁵S-methionine.

Fragments of PKH may be produced not only by recombinant production, butalso by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, supra, pp. 55-60.) Protein synthesis may be performedby manual techniques or by automation. Automated synthesis may beachieved, for example, using the ABI 431A Peptide synthesizer(Perkin-Elmer). Various fragments of PKH may be synthesized separatelyand then combined to produce the full length molecule.

THERAPEUTICS

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of PKE and various protein kinasehomologs. In addition, the expression of PKH is closely associated withcancer, reproductive tissues and hematopoietic/immune tissues.Therefore, PKH appears to play a role in cancer, autoimmune/inflammatorydisorders, and reproductive disorders. In the treatment of cancer,autoimmune/inflamratory disorders, and reproductive disorders associatedwith increased PKH expression or activity, it is desirable to decreasethe expression or activity of PKH. In the treatment of the aboveconditions associated with decreased PKH expression or activity, it isdesirable to increase the expression or activity of PKH.

Therefore, in one embodiment, PKH or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of PKH. Examples ofsuch disorders include, but are not limited to, a cancer, such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatorydisorder such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, autoimmunepolyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma.;and a reproductive disorder, such as disorders of prolactin production;infertility, including tubal disease, ovulatory defects, andendometriosis; disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, uterine fibroids, autoimmunedisorders, ectopic pregnancies, and teratogenesis; cancer of the breast,fibrocystic breast disease, and galactorrhea; disruptions ofspermatogenesis, abnormal sperm physiology, cancer of the testis, cancerof the prostate, benign prostatic hyperplasia, prostatitis, Peyronie'sdisease, impotence, carcinoma of the male breast, and gynecomastia.

In another embodiment, a vector capable of expressing PKH or a fragmentor derivative thereof may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofPKH including, but not limited to, those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified PKH in conjunction with a suitable pharmaceuticalcarrier may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of PKH including, butnot limited to, those provided above.

In still another embodiment, an agonist which modulates the activity ofPKH may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of PKH including, butnot limited to, those listed above.

In a further embodiment, an antagonist of PKH may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of PKH. Examples of such disorders include, butare not limited to, those described above. In one aspect, an antibodywhich specifically binds PKH may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express PKH.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding PKH may be administered to a subject to treat orprevent a disorder associated with increased expression or activity ofPKH including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of PKH may be produced using methods which are generallyknown in the art. In particular, purified PKH may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind PKH. Antibodies to PKH may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith PKH or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to PKH have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of PKH amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to PKH may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce PKH-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries.(See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for PKH may alsobe generated. For example, such fragments include, but are not limitedto, F(ab′)2 fragments produced by pepsin digestion of the antibodymolecule and Fab fragments generated by reducing the disulfide bridgesof the F(ab′)2 fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity. (See, e.g., Huse, W. D. et al.(1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between PKH and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering PKH epitopes is preferred, but a competitive bindingassay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for PKH. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of PKH-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple PKH epitopes, represents the average affinity,or avidity, of the antibodies for PKH. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular PKH epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which thePKH-antibody complex must withstand rigorous manipulations. Low-affinityantibody preparations with K_(a) ranging from about 10⁶ to 10⁷ L/moleare preferred for use in immunopurification and similar procedures whichultimately require dissociation of PKH, preferably in active form, fromthe antibody (Catty, D. (1988) Antibodies. Volume I: A PracticalApproach, IRL Press, Washington, D.C.; Liddell, J. E. and Cryer, A.(1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons,New York N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is preferred for use in proceduresrequiring precipitation of PKH-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encodingPKH, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingPKH may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding PKH. Thus,complementary molecules or fragments may be used to modulate PKHactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments can be designed from various locations along the codingor control regions of sequences encoding PKH.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding PKH. (See, e.g., Sambrook,supra; Ausubel, 1995, supra.)

Genes encoding PKH can be turned off by transforming a cell or tissuewith expression vectors which express high levels-of a polynucleotide,or fragment thereof, encoding PKH. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encoding PKH.Oligonucleotides derived from the transcription initiation site, e.g.,between about positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using triple helix base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature. (See, e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt.Kisco N.Y., pp. 163-177.) A complementary sequence or antisense moleculemay also be designed to block translation of mRNA by preventing thetranscript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingPKH.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding PKH. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1 997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of PKH,antibodies to PKH, and mimetics, agonists, antagonists, or inhibitors ofPKH. The compositions may be administered alone or in combination withat least one other agent, such as a stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Reminigton's Pharmaceutical Sciences (MaackPublishing, Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Saltstend to be imore soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of PKH, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example PKH or fragments thereof, antibodies of PKH, andagonists, antagonists or inhibitors of PKH, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind PKH may beused for the diagnosis of disorders characterized by expression of PKH,or in assays to monitor patients being treated with PKH or agonists,antagonists, or inhibitors of PKH. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for PKH include methods which utilizethe antibody and a label to detect PKH in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring PKH, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of PKH expression. Normal or standard values for PKHexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toPKH under conditions suitable for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of PKH expressed in subject,samples from biopsied tissues are compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding PKHmay be used for diagnostic purposes. The polynucleotides which may beused include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofPKH may be correlated with disease. The diagnostic assay may be used todetermine absence, presence, and excess expression of PKH, and tomonitor regulation of PKH levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding PKH or closely related molecules may be used to identifynucleic acid sequences which encode PKH. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding PKH, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of the PKHencoding sequences. The hybridization probes of the subject inventionmay be DNA or RNA and may be derived from the sequence of SEQ ID NO:10-18 or from genomic sequences including promoters, enhancers, andintrons of the PKH gene.

Means for producing specific hybridization probes for DNAs encoding PKHinclude the cloning of polynucleotide sequences encoding PKH or PKHderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding PKH may be used for the diagnosis ofdisorders associated with expression of PKH. Examples of such disordersinclude, but are not limited to, cancers including adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus; autoimmune/inflammatory disorders such as acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, bronchitis, cholecystitis, contact dermnatitis, Crohn'sdisease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma; and reproductive disorders includingdisorders of prolactin production; infertility, including tubal disease,ovulatory defects, and endometriosis; disruptions of the estrous cycle,disruptions of the menstrual cycle, polycystic ovary syndrome, ovarianhyperstimulation syndrome, endometrial and ovarian tumors, uterinefibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis;cancer of the breast, fibrocystic breast disease, and galactorrhea;disruptions of spermatogenesis, abnormal sperm physiology, cancer of thetestis, cancer of the prostate, benign prostatic hyperplasia,prostatitis, Peyronie's disease, impotence, carcinoma of the malebreast, and gynecomastia. The polynucleotide sequences encoding PKH maybe used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; in dipstick, pin, andmultiformat ELISA-like assays; and in microarrays utilizing fluids ortissues from patients to detect altered PKH expression. Such qualitativeor quantitative methods are well known in the art.

In aparticular aspect, the nucleotide sequences encoding PKH may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingPKH may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding PKH in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of PKH, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding PKH, under conditions suitablefor hybridization or amplification. Standard hybridization may bequantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding PKH may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding PKH, or a fragment of a polynucleotide complementary to thepolynucleotide encoding PKH, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of PKHinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;Duplaa, C. et al. (1993) Anal. Biochem. 212: 229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or calorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1 996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingPKH may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat. Genet.15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J.(1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Exanples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding PKH on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, PKH, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between PKHand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with PKH, or fragments thereof, and washed. Bound PKH is thendetected by methods well known in the art. Purified PKH can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding PKH specificallycompete with a test compound for binding PKH. In this manner, antibodiescan be used to detect the presence of any peptide which shares one ormore antigenic determinants with PKH.

In additional embodiments, the nucleotide sequences which encode PKH maybe used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I. Construction of cDNA Libraries

RNA was purchased from Clontech or isolated from tissues described inTable 4. Some tissues were homogenized and lysed in guanidiniumisothiocyanate, while others were homogenized and lysed in phenol or ina suitable mixture of denaturants, such as TRIZOL reagent (LifeTechnologies), a monophasic solution of phenol and guanidineisothiocyanate. The resulting lysates were centrifuiged over CsClcushions or extracted with chloroform. RNA was precipitated from thelysates with either isopropanol or sodium acetate and ethanol, or byother routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A+) RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Valencia Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA was isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIZAP vector system (Stratagene) orSUPERSCRIPT plasmid system (Life Technologies), using the recommendedprocedures or similar methods known in the art. (See, e.g., Ausubel,1997, supra, units 5.1-6.6). Reverse transcription was initiated usingoligo d(T) or random primers. Synthetic oligonucleotide adapters wereligated to double stranded cDNA, and the cDNA was digested with theappropriate restriction enzyme or enzymes. For most libraries, the cDNAwas size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B,or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (LifeTechnologies), or pINCY (Incyte Pharmaceuticals, Palo Alto Calif.).Recombinant plasmids were transformed into competent E. coli cellsincluding XL1-Blue, XL1-Blue MRF, or SOLR from Stratagene or DH5α,DH10B, or ELECTROMAX DH10B from Life Technologies.

II. Isolation of cDNA Clones

Plasmids were recovered from host cells by in vivo excision, using theUNIZAP vector system (Stratagene) or cell lysis. Plasmids were purifiedusing at least one of the following: a Magic or WIZARD Minipreps DNApurification system (Promega); an AGTC Miniprep purification kit (EdgeBiosystems, Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 PlusPlasmid, QIAWELL 8 Ultra. plasmid purification systems or the R.E.A.L.Prep 96 plasmid kit from QIAGEN. Following precipitation, plasmids wereresuspended in 0.1 ml of distilled water and stored, with or withoutlyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V.B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Ore.) and a Fluoroskan II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

The-cDNAs were prepared for sequencing using the ABI CATALYST 800(Perkin-Elmer) or the HYDRA microdispenser (Robbins Scientific) orMICROLAB 2200 (Hamilton) systems in combination with the PTC-200 thermalcyclers (MJ Research). The cDNAs were sequenced using the ABI PRISM 373or 377 sequencing systems (Perkin-Elmer) and standard ABI protocols,base calling software, and kits. In one alternative, cDNAs weresequenced using the MEGABACE 1000 DNA sequencing system (MolecularDynamics). In another alternative, the cDNAs were amplified andsequenced using the ABI PRISM BIGDYE Terminator cycle sequencing readyreaction kit (Perkin-Elmer). In yet another alternative, cDNAs weresequenced using solutions and dyes from Amersham Pharmacia Biotech.Reading frames for the ESTs were determined using standard methods(reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequenceswere selected for extension using the techniques disclosed in Example V.

The polynucleotide sequences derived from cDNA, extension, and shotgunsequencing were assembled and analyzed using a combination of softwareprograms which utilize algorithms well known to those skilled in theart. Table 5 summarizes the software programs, descriptions, references,and threshold parameters used. The first column of Table 5 shows thetools, programs, and algorithms used, the second column provides a briefdescription thereof, the third column presents the references which areincorporated by reference herein, and the fourth column presents, whereapplicable, the scores, probability values, and other parameters used toevaluate the strength of a match between two sequences (the higher theprobability the greater the homology). Sequences were analyzed usingMACDNASIS PRO software (Hitachi Software Engineering, South SanFrancisco Calif.) and LASERGENE software (DNASTAR).

The polynucleotide sequences were validated by removing vector, linker,and polyA sequences and by masking ambiguous bases, using algorithms andprograms based on BLAST, dynamic programing, and dinucleotide nearestneighbor analysis. The sequences were then queried against a selectionof public databases such as GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS to acquire annotation,using programs based on BLAST, FASTA, and BLIMPS. The sequences wereassembled into full length polynucleotide sequences using programs basedon Phred, Phrap, and Consed, and were screened for open reading framesusing programs based on GeneMark, BLAST, and FASTA. The full lengthpolynucleotide sequences were translated to derive the correspondingfull length amino acid sequences, and these full length sequences weresubsequently analyzed by querying against databases such as the GenBankdatabases (described above), SwissProt, BLOCKS, PRINTS, PFAM, andProsite.

The programs described above for the assembly and analysis of fulllength polynucleotide and amino acid sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO: 10-18.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies were described in TheInvention section above.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel, 1995, supra, ch.4 and 16.)

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ database (Incyte Pharmaceuticals). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or similar. The basis ofthe search is the product score, which is defined as:$\frac{\% \quad {sequence}\quad {identity}\quad \times \quad {maximum}\quad {BLAST}\quad {score}}{100}$

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact. Similarmolecules are usually identified by selecting those which show productscores between 15 and 40, although lower scores may identify relatedmolecules.

The results of northern analyses are reported as a percentagedistribution of libraries in which the transcript encoding PKH occurred.Analysis involved the categorization of cDNA libraries by organ/tissueand disease. The organ/tissue categories included cardiovascular,dermatologic, developmental, endocrine, gastrointestinal,hematopoietic/immune, musculoskeletal, nervous, reproductive, andurologic. The disease/condition categories included cancer,inflammation/trauma, cell proliferation, neurological, and pooled. Foreach category, the number of libraries expressing the sequence ofinterest was counted and divided by the total number of libraries acrossall categories. Percentage values of tissue-specific and disease- orcondition-specific expression are reported in Table 3.

V. Extension of PKH Encoding Polynucleotides

The full length nucleic acid sequence of SEQ ID NO: 10-18 was producedby extension of an appropriate fragment of the full length moleculeusing oligonucleotide primers designed from this fragment. One primerwas synthesized to initiate 5′ extension of the known fragment, and theother primer, to initiate 3′ extension of the known fragment. Theinitial primers were designed using OLIGO 4.06 software (NationalBiosciences), or another appropriate program, to be about 22 to 30nucleotides in length, to have a GC content of about 50% or more, and toanneal to the target sequence at temperatures of about 68° C. to about72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 nmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and β-mercaptoethanol, Taq DNA polymerase (Amersham PharmaciaBiotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

The concentration of DNA in each well was determined by dispensing 100μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; MolecularProbes, Eugene Oreg.) dissolved in 1X TE and 0.5 μl of undiluted PCRproduct into each well of an opaque fluorimeter plate (Coming Costar,Acton Mass.), allowing the DNA to bind to the reagent. The plate wasscanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measurethe fluorescence of the sample and to quantify the concentration of DNA.A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1% agarose mini-gel to determine which reactionswere successfutl in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Pharmacia Biotech). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase(Stratagene) to fill-in restriction site overhangs, and transfected intocompetent E. coli cells. Transformed cells were selected onantibiotic-containing media, individual colonies were picked andcultured overnight at 37° C. in 384-well plates in LB/2×carb liquidmedia.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and step 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethysulphoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Perkin-Elmer).

In like manner, the nucleotide sequence of SEQ ID NO: 10-18 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for such extension, and an appropriate genomiclibrary.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO: 10-18 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [λ-³²P] adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, XbaI,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT-AR film(Eastman Kodak, Rochester N.Y.) is exposed to the blots hybridizationpatterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragmentsthereof corresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking, followed by thermal and chemical treatments andsubsequent drying. (See, e.g., Schena, M. et al. (1995) Science270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.)Fluorescent probes are prepared and used for hybridization to theelements on the substrate. The substrate is analyzed by proceduresdescribed above.

VIII. Complementary Polynucleotides

Sequences complementary to the PKH-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring PKH. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of PKH. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the PKH-encoding transcript.

IX. Expression of PKH

Expression and purification of PKH is achieved using bacterial orvirus-based expression systems. For expression of PKH in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21 (DE3). Antibiotic resistant bacteria express PKH uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof PKH in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding PKH by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, PKH is synthesized as a fusion protein with,e.g., glutathione S-transferase (GST) or a peptide epitope tag, such asFLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma jaronicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamPharmacia Biotech). Following purification, the GST moiety can beproteolytically cleaved from PKH at specifically engineered sites. FLAG,an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch 10 and 16). Purified PKH obtained by these methods can be useddirectly in the following activity assay.

X. Demonstration of PKH Activity

An assay for PKH activity measures the phosphorylation of a substrate inthe presence of gamma-labeled ³²P-ATP. PKH is incubated with anappropriate substrate and ³²P-ATP in a buffered solution. ³²P-labeledproduct is separated from free ³²P-ATP by gel electrophoresis orchromatographic procedures, and the incorporated ³²P is quantified byphosphoimage analysis or scintillation counter. The amount of ³²Pdetected is proportional to the activity of PKH in this assay. Thespecific amino acid residue phosphorylated by PKH may be determined byphosphoamino acid analysis of the labeled, hydrolyzed protein.

XI. Functional Assays

PKH function is assessed by expressing the sequences encoding PKH atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude PCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, CarlsbadCalif.), both of which contain the cytomegalovirus promoter. 5-10 μg ofrecombinant vector are transiently transfected into a human cell line,preferably of endothelial or hematopoietic origin, using either liposomeformulations or electroporation. 1-2 μg of an additional plasmidcontaining sequences encoding a marker protein are co-transfected.Expression of a marker protein provides a means to distinquishtransfected cells from nontransfected cells and is a reliable predictorof cDNA expression from the recombinant vector. Marker proteins ofchoice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64,or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laseroptics-based technique, is used to identify transfected cells expressingGFP or CD64-GFP, and to evaluate properties, for example, theirapoptotic state. FCM detects and quantifies the uptake of fluorescentmolecules that diagnose events preceding or coincident with cell death.These events include changes in nuclear DNA content as measured bystaining of DNA with propidium iodide; changes in cell size andgranularity as measured by forward light scatter and 90 degree sidelight scatter; down-regulation of DNA synthesis as measured by decreasein bromodeoxyuridine uptake; alterations in expression of cell surfaceand intracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cytometgy, Oxford, New York N.Y.

The influence of PKH on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding PKHand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding PKH and other genes of interest canbe analyzed by northern analysis or microarray techniques.

XII. Production of PKH Specific Antibodies

PKH substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the PKH amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubel,1995, supra, ch. 11.)

Typically, oligopeptides 15 residues in length are synthesized using anABI 431A Peptide synthesizer (Perkin-Elmer) using fmoc-chemistry andcoupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide activity by, for example,binding the peptide to plastic, blocking with 1% BSA, reacting withrabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

XIII. Purification of Naturally Occurring PKH Using Specific Antibodies

Naturally occurring or recombinant PKH is substantially purified byimmunoaffinity chromatography using antibodies specific for PKH. Animmunoaffinity column is constructed by covalently coupling anti-PKHantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharnacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing PKH are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof PKH (e.g., high ionic strength buffers in the presence of detergent).The column is eluted under conditions that disrupt antibody/PKH binding(e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope,such as urea or thiocyanate ion), and PKH is collected.

XIV. Identification of Molecules Which Interact with PKH

PKH, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled PKH, washed, and anywells with labeled PKH complex are assayed. Data obtained usingdifferent concentrations of PKH are used to calculate values for thenumber, affinity, and association of PKH with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

TABLE 1 Amino Acid Nucleotide SEQ ID NO: SEQ ID NO: Clone ID LibraryFragments 1 10  119819 MUSCNOT01 119819H1 (MUSCNOT01), 1476434H1(CORPNOT02), 1719355H1 (BLADNOT06), 3016364F6 (MUSCNOT07), 3373249F6(CONNTUT05) 2 11  132750 BMARNOT02 132750H1 and 132750X335D1(BMARNOT02), 287300R1 (EOSIHET02), 1271292F1 (TESTTUT02), 3343321H1(SPLNNOT09) 3 12  507669 TMLR3DT01 101010R6 (ADRENOT01), 507669H1 and507669R6 (TMLR3DT01), 697674H1 (SYNORAT03), 851624T1 (NGANNOT01),1270195F7 (BRAINOT09) 4 13 1439938 THYRNOT03 814363X11 (OVARTUT01),822246X13 (KERANOT02), 1439938H1 (THYRNOT03), 3365318F6 (PROSBPT02) 5 141447427 PLACNOT02 712366R6 (SYNORAT04), 1447427H1 (PLACNOT02), 2179842F6(SININOT01), 2697460F6 (UTRSNOT12) 6 15 1567782 UTRSNOT05 1567782H1 and1567786F6 (UTRSNOT05), 2289257X42F1 (BRAINON01), 2839231T6 (DRGLNOT01),SAFB00259F1 7 16 2295842 BRSTNOT05 606173H1 (BRSTTUT01), 865560R1(BRAITUT03), 1441018F6 (THYRNOT03), 1706132T6 (DUODNOT02), 1811824F6(PROSTUT12), 2223155H1 (LUNGNOT18), 2295842H1 (BRSTNOT05) 8 17 2605059LUNGTUT07 381188R6 (HYPONOB01), 381188T6 (HYPONOB01), 691185T6(LUNGTUT02), 1824201F6 (GBLATUT01), 2605059F6, 2605059H1 and2605059X304F1 (LUNGTUT07) 9 18 3000825 TLYMNOT06 2015630F6 (ADRENOT07),3000825F6 and 3000825H1 (TLYMNOT06)

TABLE 2 Amino Acid Amino Acid Potential Signature Seq ID NO: ResiduesPhosphorylation Sites Sequence Identification Analytical Methods 1 297S285 T29 S197 S202 S225 A117-L295 SR protein- PFAM, BLAST, BLOCKS, S285specific kinase MOTIFS 2 287 S283 T106 T179 S194 S31-T284 protein kinasePFAM, BLOCKS, PRINTS, S274 T69 T184 S233 S270 homolog BLAST, MOTIFS 3346 S7 T105 S160 T232 T244 E125-D333 tyrosine kinase PFAM, BLAST, T282S329 T336 S214 BLOCKS, PRINTS, T255 Y231 PROFILESCAN, MOTIFS 4  90 S60S9 S62 F28-R78 protein kinase PFAM, BLOCKS, BLAST, homolog MOTIFS 5 327S96 T210 S277 T40 S137 Y11-N235 protein kinase PFAM, BLOCKS, PRINTS,S179 T273 Y178 Y310 homolog BLAST, MOTIFS 6 345 S327 S23 S41 S48 S123Y4-I226 serine/threonine- PFAM, BLAST, BLOCKS, S219 T319 T166 S175 Y30and tyrosine- PRINTS, PROFILESCAN, specific protein MOTIFS kinase, Nek17 424 T97 S218 S298 T389 S413 D202-V412 protein kinase PFAM, BLOCKS,PRINTS, T54 T62 T89 T109 S112 homolog BLAST, MOTIFS S151 S223 S229 S286S318 8  99 cdc2+/CDC28- BLAST related protein kinase 9 138 T91 T24 T57T91 T14 R72-V101 serine/threonine PFAM, BLAST, MOTIFS protein kinase

TABLE 3 Nucleotide Disease or Condition Seq ID NO: Tissue Expression(Fraction of Total) (Fraction of Total) Vector 10 Nervous (0.400)Musculoskeletal (0.200) Cancer (0.500) Neurological (0.300) pBluescriptCardiovascular (0.100) 11 Reproductive (0.316) Hematopoietic/ImmuneInflammation (0.421) Cancer (0.368) pBluescript (0.211) Gastrointestinal(0.158) 12 Hematopoietic/Immune (0.514) Gastrointestinal Inflammation(0.595) Cancer (0.243) pBluescript (0.189) Reproductive (0.081) 13Reproductive (0.375) Developmental (0.125) Cancer (0.375) Inflammation(0.250) pINCY Endocrine (0.125) 14 Reproductive (0.346) Nervous (0.269)Cancer (0.462) Inflammation (0.385) pINCY Hematopoietic/Immune (0.231)15 Nervous (0.500) Developmental (0.167) Cancer (0.833) Inflammation(0.333) pINCY Musculoskeletal (0.167) 16 Reproductive (0.290)Gastrointestinal (0.145) Cancer (0.420) Inflammation (0.362) pSPORT1Nervous (0.130) 17 Nervous (0.250) Gastrointestinal (0.167) Cancer(0.500) Inflammation (0.250) pINCY Hematopoietic/Immune (0.167) 18Endocrine (0.333) Hematopoietic/Immune (0.333) Inflammation (0.667)Cancer (0.333) pINCY Reproductive (0.333)

TABLE 4 Nucleo- tide SEQ ID NO: Library Library Comment 10 MUSCNOT01Library was constructed at Stratagene (STR937209), using RNA isolatedfrom the skeletal muscle tissue of a patient with malignanthyperthermia. 11 BMARNOT02 Library was constructed using RNA isolatedfrom the bone marrow of 24 male and female Caucasian donors, 16 to 70years old. (RNA came from Clontech.) 12 TMLR3DT02 Library wasconstructed using RNA isolated from non-adherent peripheral blood mono-nuclear cells collected from a pool of male and female donors. Cellsfrom each donor were purified on Ficoll Hypaque, then co-cultured for 72hours. The cells were pooled, washed once in PBS, lysed in a buffercontaining GuSCN, and spun through CsC1 to obtain RNA. PolyA RNA wasisolated using a Qiagen Oligotex kit. 13 THYRNOT03 Library wasconstructed using RNA isolated from thyroid tissue removed from the leftthyroid of a 28-year-old Caucasian female during a completethyroidectomy. Pathology indicated a small nodule of adenomatoushyperplasia present in the left thyroid. Pathology for the associatedtumor tissue indicated dominant follicular adenoma, forming awell-encapsulated mass in the left thyroid. 14 PLACNOT02 Library wasconstructed using RNA isolated from the placental tissue of a Hispanicfemale fetus, who was prematurely delivered at 21 weeks' gestation.Serologies of the mother's blood were positive for cytomegalovirus. 15UTRSNOT05 Library was constructed using RNA isolated from the uterinetissue of a 45-year-old Caucasian female during a total abdominalhysterectomy and total colectomy. Pathology for the associated tumortissue indicated multiple leiomyomas of the myometrium. 16 BRSTNOT05Library was constructed using RNA isolated from breast tissue removedfrom a 58-year-old Caucasian female during a unilateral extended simplemastectomy. Pathology for the associated tumor tissue indicatedmulticentric invasive grade 4 lobular carcinoma. Family history includedbreast and prostate cancer. 17 LUNCTUT07 Library was constructed usingRNA isolated from lung tumor tissue removed from the upper lobe of a50-year-old Caucasian male during segmental lung resection. Pathologyindicated an invasive grade 4 squamous cell adeno- carcinoma. Patienthistory included tobacco use. Family history included skin cancer. 18TLYMNOT06 Library was constructed using RNA isolated from activated Th2cells. These cells were differentiated from umbilical cord CD4 T cellswith IL-4 in the presence of anti- IL-12 antibodies and B7-transfectedCOS cells, and then activated for six hours with anti- CD3 and anti-CD28antibodies.

TABLE 5 Program Description Reference Parameter Threshold ABI FACTURA Aprogram that removes vector sequences and masks Perkin-Elmer AppliedBiosystems, ambiguous bases in nucleic acid sequences. Foster City, CA.ABI/ A Fast Data Finder useful in comparing and annotating Perkin-ElmerApplied Biosystems, Mismatch <50% PARACEL FDF amino acid or nucleic acidsequences. Foster City, CA; Paracel Inc., Pasadena, CA. ABI A programthat assembles nucleic acid sequences. Perkin-Elmer Applied Biosystems,AutoAssembler Foster City, CA. BLAST A Basic Local Alignment Search Tooluseful in sequence Altschul, S. F. et al. (1990) J. Mol. Biol. ESTs:Probability similarity search for amino acid and nucleic acid215:403-410; Altschul, S. F. et al. (1997) value = 1.0E-8 sequences.BLAST includes five functions: blastp, blastn, Nucleic Acids Res.25:3389-3402. or less blastx, tblastn, and tblastx. Full Lengthsequences: Probability value = 1.0E-10 or less FASTA A Pearson andLipman algorithm that searches for Pearson, W. R. and D. J. Lipman(1988) Proc. ESTs: fasta E similarity between a query sequence and agroup of Natl. Acad Sci. 85:2444-2448; Pearson, W. R. value = 1.06E-6sequences of the same type. FASTA comprises as least (1990) MethodsEnzymol. 183:63-98; and Assembled ESTs: five functions: fasta, tfasta,fastx, tfastx, and ssearch. Smith, T. F. and M. S. Waterman (1981) Adv.fasta Identity = Appl. Math. 2:482-489. 95% or greater and Match length= 200 bases or greater; fastx E value = 1.0E-8 or less Full Lengthsequences: fastx score = 100 or greater BLIMPS A BLocks IMProvedSearcher that matches a sequence Henikoff S and J. G. Henikoff, Nucl.Acid Res., Score = 1000 or against those in BLOCKS and PRINTS databasesto 19:6565-72, 1991. J. G. Henikoff and S. greater; Ratio of search forgene families, sequence homology, and Henikoff (1996) Methods Enzymol.266:88-105; Score/Strength = structural fingerprint regions. andAttwood, T. K. et al. (1997) J. Chem. Inf. 0.75 or larger; and Comput.Sci. 37:417-424. Probability value = 1.0E-3 or less PFAM A Hidden MarkovModels-based application useful for Krogh, A. et al. (1994) J. Mol.Biol., 235:1501- Score = 10-50 bits, protein family search. 1531;Sonnhammer, E. L. L. etal. (1988) depending on Nucleic Acids Res.26:320-322. individual protein families ProfileScan An algorithm thatsearches for structural and sequence Gribskov, M. et al. (1988) CABIOS4:61-66; Score = 4.0 or motifs in protein sequences that match sequenceGribskov, et al. (1989) Methods Enzymol. greater patterns defined inProsite. 183:146-159; Bairoch, A. et al. (1997) Nucleic Acids Res.25:217-221. Phred A base-calling algorithm that examines automatedEwing, B. et al. (1998) Genome sequencer traces with high sensitivityand probability. Res. 8:175-185; Ewing, B. and P. Green (1998) GenomeRes. 8:186- 194. Phrap A Phils Revised Assembly Program including SWATand Smith, T. F. and M. S. Waterman (1981) Adv. Score = 120 orCrossMatch, programs based on efficient implementation Appl. Math.2:482-489; Smith, T. F. and M. S. greater; Match of the Smith-Watermanalgorithm, useful in searching Waterman (1981) J. Mol. Biol.147:195-197; length = 56 or sequence homology and assembling DNAsequences. and Green, P., University of Washington, greater Seattle, WA.Consed A graphical tool for viewing and editing Phrap Gordon, D. et al.(1998) Genome assemblies Res.8:195-202. SPScan A weight matrix analysisprogram that scans protein Nielson, H. et al. (1997) Protein EngineeringScore = 5 or sequences for the presence of secretory signal peptides.10:1-6; Claverie, J. M. and S. Audic (1997) greater CABIOS 12:431-439.Motifs A program that searches amino acid sequences for Bairoch et al.supra; Wisconsin patterns that matched those defined in Prosite. PackageProgram Manual, version 9, page M51-59, Genetics Computer Group,Madison, WI.

18 1 297 PRT Homo sapiens 119819 1 Met Arg Arg Lys Arg Lys Gln Gln LysArg Leu Leu Glu Glu Arg 1 5 10 15 Leu Arg Asp Leu Gln Arg Leu Glu AlaMet Glu Ala Ala Thr Gln 20 25 30 Ala Glu Asp Ser Gly Leu Arg Leu Asp GlyGly Ser Gly Ser Thr 35 40 45 Ser Ser Ser Gly Cys His Pro Gly Gly Ala ArgAla Gly Pro Ser 50 55 60 Pro Ala Ser Ser Ser Pro Ala Pro Gly Gly Gly ArgSer Leu Ser 65 70 75 Ala Gly Ser Gln Thr Ser Gly Phe Ser Gly Ser Leu PheSer Pro 80 85 90 Ala Ser Cys Ser Ile Leu Ser Gly Ser Ser Asn Gln Arg GluThr 95 100 105 Gly Gly Leu Leu Ser Pro Ser Thr Pro Phe Gly Ala Ser AsnLeu 110 115 120 Leu Val Asn Pro Leu Glu Pro Gln Asn Ala Asp Lys Ile LysIle 125 130 135 Lys Ile Ala Asp Leu Gly Asn Ala Cys Trp Val His Lys HisPhe 140 145 150 Thr Glu Asp Ile Gln Thr Arg Gln Tyr Arg Ala Val Glu ValLeu 155 160 165 Ile Gly Ala Glu Tyr Gly Pro Pro Ala Asp Ile Trp Ser ThrAla 170 175 180 Cys Met Ala Phe Glu Leu Ala Thr Gly Asp Tyr Leu Phe GluPro 185 190 195 His Ser Gly Glu Asp Tyr Ser Arg Asp Glu Asp His Ile AlaHis 200 205 210 Ile Val Glu Leu Leu Gly Asp Ile Pro Pro Ala Phe Ala LeuSer 215 220 225 Gly Arg Tyr Ser Arg Glu Phe Phe Asn Arg Arg Gly Glu LeuArg 230 235 240 His Ile His Asn Leu Lys His Trp Gly Leu Tyr Glu Val LeuMet 245 250 255 Glu Lys Tyr Glu Trp Pro Leu Glu Gln Ala Thr Gln Phe SerAla 260 265 270 Phe Leu Leu Pro Met Asn Glu Tyr Ile Pro Glu Lys Arg AlaSer 275 280 285 Ala Arg Asp Cys Leu Gln His Pro Trp Leu Gln Pro 290 2952 287 PRT Homo sapiens 132750 2 Met Gln Glu Ile Pro Gln Glu Gln Ile LysGlu Ile Lys Lys Glu 1 5 10 15 Gln Leu Ser Gly Ser Pro Trp Ile Leu LeuArg Glu Asn Glu Val 20 25 30 Ser Thr Leu Tyr Lys Gly Glu Tyr His Arg AlaPro Val Ala Ile 35 40 45 Lys Val Phe Lys Lys Leu Gln Ala Gly Ser Ile AlaIle Val Arg 50 55 60 Gln Thr Phe Asn Lys Glu Ile Lys Thr Met Lys Lys PheGlu Ser 65 70 75 Pro Asn Ile Leu Arg Ile Phe Gly Ile Cys Ile Asp Glu ThrVal 80 85 90 Thr Pro Pro Gln Phe Ser Ile Val Met Glu Tyr Cys Glu Leu Gly95 100 105 Thr Leu Arg Glu Leu Leu Asp Arg Glu Lys Asp Leu Thr Leu Gly110 115 120 Lys Arg Met Val Leu Val Leu Gly Ala Ala Arg Gly Leu Tyr Arg125 130 135 Leu His His Ser Glu Ala Pro Glu Leu His Gly Lys Ile Arg Ser140 145 150 Ser Asn Phe Leu Val Thr Gln Gly Tyr Gln Val Lys Leu Ala Gly155 160 165 Phe Glu Leu Arg Lys Thr Gln Thr Ser Met Ser Leu Gly Thr Thr170 175 180 Arg Glu Lys Thr Asp Arg Val Lys Ser Thr Ala Tyr Leu Ser Pro185 190 195 Gln Glu Leu Glu Asp Val Phe Tyr Gln Tyr Asp Val Lys Ser Glu200 205 210 Ile Tyr Ser Phe Gly Ile Val Leu Trp Glu Ile Ala Thr Gly Asp215 220 225 Ile Pro Phe Gln Gly Cys Asn Ser Glu Lys Ile Arg Lys Leu Val230 235 240 Ala Val Lys Arg Gln Gln Glu Pro Leu Gly Glu Asp Cys Pro Ser245 250 255 Glu Leu Arg Glu Ile Ile Asp Glu Cys Arg Ala His Asp Pro Ser260 265 270 Val Arg Pro Ser Val Asp Glu Ile Leu Lys Lys Leu Ser Thr Phe275 280 285 Ser Lys 3 346 PRT Homo sapiens 507669 3 Met Gly Cys Gly CysSer Ser His Pro Glu Asp Asp Trp Met Glu 1 5 10 15 Asn Ile Asp Val CysGlu Asn Cys His Tyr Pro Ile Val Pro Leu 20 25 30 Asp Gly Lys Gly Thr LeuLeu Ile Arg Asn Gly Ser Glu Val Arg 35 40 45 Asp Pro Leu Val Thr Tyr GluGly Ser Asn Pro Pro Ala Ser Pro 50 55 60 Leu Gln Asp Asn Leu Val Ile AlaLeu His Ser Tyr Glu Pro Ser 65 70 75 His Asp Gly Asp Leu Gly Phe Glu LysGly Glu Gln Leu Arg Ile 80 85 90 Leu Glu Gln Ser Gly Glu Trp Trp Lys AlaGln Ser Leu Thr Thr 95 100 105 Gly Gln Glu Gly Phe Ile Pro Phe Asn PheVal Ala Lys Ala Asn 110 115 120 Ser Leu Glu Pro Glu Ala Asn Leu Met LysGln Leu Gln His Gln 125 130 135 Arg Leu Val Arg Leu Tyr Ala Val Val ThrGln Glu Pro Ile Tyr 140 145 150 Ile Ile Thr Glu Tyr Met Glu Asn Gly SerLeu Val Asp Phe Leu 155 160 165 Lys Thr Pro Ser Gly Ile Lys Leu Thr IleAsn Lys Leu Leu Asp 170 175 180 Met Ala Ala Gln Ile Ala Glu Gly Met AlaPhe Ile Glu Glu Arg 185 190 195 Asn Tyr Ile His Arg Asp Leu Arg Ala AlaAsn Ile Leu Val Ser 200 205 210 Asp Thr Leu Ser Cys Lys Ile Ala Asp PheGly Leu Ala Arg Leu 215 220 225 Ile Glu Asp Asn Glu Tyr Thr Ala Arg GluGly Ala Lys Phe Pro 230 235 240 Ile Lys Trp Thr Ala Pro Glu Ala Ile AsnTyr Gly Thr Phe Thr 245 250 255 Ile Lys Ser Asp Val Trp Ser Phe Gly IleLeu Leu Thr Glu Ile 260 265 270 Val Thr His Gly Arg Ile Pro Tyr Pro GlyMet Thr Asn Pro Glu 275 280 285 Val Ile Gln Asn Leu Glu Arg Gly Tyr ArgMet Val Arg Pro Asp 290 295 300 Asn Cys Pro Glu Glu Leu Tyr Gln Leu MetArg Leu Cys Trp Lys 305 310 315 Glu Arg Pro Glu Asp Arg Pro Thr Phe AspTyr Leu Arg Ser Val 320 325 330 Leu Glu Asp Phe Phe Thr Ala Thr Glu GlyGln Tyr Gln Pro Gln 335 340 345 Pro 4 90 PRT Homo sapiens 1439938 4 MetPro Ala Gly Gly Arg Ala Gly Ser Leu Lys Asp Pro Asp Val 1 5 10 15 AlaGlu Leu Phe Phe Lys Asp Asp Pro Glu Lys Leu Phe Ser Asp 20 25 30 Leu ArgGlu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala 35 40 45 Arg Asp ValArg Asn Ser Glu Val Val Ala Ile Lys Lys Met Ser 50 55 60 Tyr Ser Gly LysGln Ser Asn Glu Lys Trp Gln Asp Ile Ile Lys 65 70 75 Glu Val Arg Arg ArgArg Arg Val Gly Arg Glu Asp Glu Glu Arg 80 85 90 5 327 PRT Homo sapiens1447427 5 Met Ser Ser Phe Leu Pro Glu Gly Gly Cys Tyr Glu Leu Leu Thr 15 10 15 Val Ile Gly Lys Gly Phe Glu Asp Leu Met Thr Val Asn Leu Ala 2025 30 Arg Tyr Lys Pro Thr Gly Glu Tyr Val Thr Val Arg Arg Ile Asn 35 4045 Leu Glu Ala Cys Ser Asn Glu Met Val Thr Phe Leu Gln Gly Glu 50 55 60Leu His Val Ser Lys Leu Phe Asn His Pro Asn Ile Val Pro Tyr 65 70 75 ArgAla Thr Phe Ile Ala Asp Asn Glu Leu Trp Val Val Thr Ser 80 85 90 Phe MetAla Tyr Gly Ser Ala Lys Asp Leu Ile Cys Thr His Phe 95 100 105 Met AspGly Met Asn Glu Leu Ala Ile Ala Tyr Ile Leu Gln Gly 110 115 120 Val LeuLys Ala Leu Asp Tyr Ile His His Met Gly Tyr Val His 125 130 135 Arg SerVal Lys Ala Ser His Ile Leu Ile Ser Val Asp Gly Lys 140 145 150 Val TyrLeu Ser Gly Leu Arg Thr Thr Leu Ser Met Ile Ser His 155 160 165 Gly GlnArg Gln Arg Val Val His Asp Phe Pro Lys Tyr Ser Val 170 175 180 Lys ValLeu Pro Trp Leu Ser Pro Glu Val Leu Gln Gln Asn Leu 185 190 195 Gln GlyTyr Asp Ala Lys Ser Asp Ile Tyr Ser Val Gly Ile Thr 200 205 210 Ala CysGlu Leu Ala Asn Gly His Val Pro Phe Lys Asp Met Pro 215 220 225 Ala ThrGln Met Leu Leu Glu Lys Leu Asn Gly Thr Val Pro Cys 230 235 240 Leu LeuAsp Thr Ser Thr Ile Pro Ala Glu Glu Leu Thr Met Ser 245 250 255 Pro SerArg Ser Val Ala Asn Ser Gly Leu Ser Asp Ser Leu Thr 260 265 270 Thr SerThr Pro Arg Pro Ser Asn Gly Asp Ser Pro Ser His Pro 275 280 285 Tyr HisArg Thr Phe Ser Pro His Phe His His Phe Val Glu Gln 290 295 300 Cys LeuGln Arg Asn Pro Asp Ala Arg Tyr Pro Cys Trp Pro Gly 305 310 315 Pro GlyLeu Arg Glu Ser Arg Gly Cys Ser Gly Gly 320 325 6 345 PRT Homo sapiens1567782 6 Met Glu Lys Tyr Val Arg Leu Gln Lys Ile Gly Glu Gly Ser Phe 15 10 15 Gly Lys Ala Ile Leu Val Lys Ser Thr Glu Asp Gly Arg Gln Tyr 2025 30 Val Ile Lys Glu Ile Asn Ile Ser Arg Met Ser Ser Lys Glu Arg 35 4045 Glu Glu Ser Arg Arg Glu Val Ala Val Leu Ala Asn Met Lys His 50 55 60Pro Asn Ile Val Gln Tyr Arg Glu Ser Phe Glu Gly Ile Leu Asp 65 70 75 TrpPhe Val Gln Ile Cys Leu Ala Leu Lys His Val His Asp Arg 80 85 90 Lys IleLeu His Arg Asp Ile Lys Ser Gln Asn Ile Phe Leu Thr 95 100 105 Lys AspGly Thr Val Gln Leu Gly Asp Phe Gly Ile Ala Arg Val 110 115 120 Leu AsnSer Thr Val Glu Leu Ala Arg Thr Cys Ile Gly Thr Pro 125 130 135 Tyr TyrLeu Ser Pro Glu Ile Cys Glu Asn Lys Pro Tyr Asn Asn 140 145 150 Lys SerAsp Ile Trp Ala Leu Gly Cys Val Leu Tyr Glu Leu Cys 155 160 165 Thr LeuLys His Ala Phe Glu Ala Gly Ser Met Lys Asn Leu Val 170 175 180 Leu LysIle Ile Ser Gly Ser Phe Pro Pro Val Ser Leu His Tyr 185 190 195 Ser TyrAsp Leu Arg Ser Leu Val Ser Gln Leu Phe Lys Arg Asn 200 205 210 Pro ArgAsp Arg Pro Ser Val Asn Ser Ile Leu Glu Lys Gly Phe 215 220 225 Ile AlaLys Arg Ile Glu Lys Phe Leu Ser Pro Gln Leu Ile Ala 230 235 240 Glu GluPhe Cys Leu Lys Thr Phe Ser Lys Phe Gly Ser Gln Pro 245 250 255 Ile ProAla Lys Arg Pro Ala Ser Gly Gln Asn Ser Ile Ser Val 260 265 270 Met ProAla Gln Lys Ile Thr Lys Pro Ala Ala Lys Tyr Gly Ile 275 280 285 Pro LeuAla Tyr Lys Lys Tyr Gly Asp Lys Lys Leu His Glu Lys 290 295 300 Lys ProLeu Gln Lys His Lys Gln Ala His Gln Thr Pro Glu Lys 305 310 315 Arg ValAsn Thr Gly Glu Glu Arg Arg Lys Ile Ser Glu Glu Ala 320 325 330 Ala ArgLys Arg Arg Leu Glu Phe Ile Glu Lys Asp Lys Glu Arg 335 340 345 7 424PRT Homo sapiens 2295842 7 Met Ile Ser Phe Cys Pro Asp Cys Gly Lys SerIle Gln Ala Ala 1 5 10 15 Phe Lys Phe Cys Pro Tyr Cys Gly Asn Ser LeuPro Val Glu Glu 20 25 30 His Val Gly Ser Gln Thr Phe Val Asn Pro His ValSer Ser Phe 35 40 45 Gln Gly Ser Gly Ser Arg Pro Pro Thr Pro Lys Ser SerPro Gln 50 55 60 Lys Thr Arg Lys Ser Pro Gln Val Thr Arg Gly Ser Pro GlnLys 65 70 75 Thr Ser Cys Ser Pro Gln Lys Thr Arg Gln Ser Pro Gln Thr Leu80 85 90 Lys Arg Ser Arg Val Thr Thr Ser Leu Glu Ala Leu Pro Thr Gly 95100 105 Thr Val Leu Thr Asp Lys Ser Gly Arg Gln Trp Lys Leu Lys Ser 110115 120 Phe Gln Thr Arg Asp Asn Gln Gly Ile Leu Tyr Glu Ala Ala Pro 125130 135 Thr Ser Thr Leu Thr Cys Asp Ser Gly Pro Gln Lys Gln Lys Phe 140145 150 Ser Leu Lys Leu Asp Ala Lys Asp Gly Arg Leu Phe Asn Glu Gln 155160 165 Asn Phe Phe Gln Arg Ala Ala Lys Pro Leu Gln Val Asn Lys Trp 170175 180 Lys Lys Leu Tyr Ser Thr Pro Leu Leu Ala Ile Pro Thr Cys Met 185190 195 Gly Phe Gly Val His Gln Asp Lys Tyr Arg Phe Leu Val Leu Pro 200205 210 Ser Leu Gly Arg Ser Leu Gln Ser Ala Leu Asp Val Ser Pro Lys 215220 225 His Val Leu Ser Glu Arg Ser Val Leu Gln Val Ala Cys Arg Leu 230235 240 Leu Asp Ala Leu Glu Phe Leu His Glu Asn Glu Tyr Val His Gly 245250 255 Asn Val Thr Ala Glu Asn Ile Phe Val Asp Pro Glu Asp Gln Ser 260265 270 Gln Val Thr Leu Ala Gly Tyr Gly Phe Ala Phe Arg Tyr Cys Pro 275280 285 Ser Gly Lys His Val Ala Tyr Val Glu Gly Ser Arg Ser Pro His 290295 300 Glu Gly Asp Leu Glu Phe Ile Ser Met Asp Leu His Lys Gly Cys 305310 315 Gly Pro Ser Arg Arg Ser Asp Leu Gln Ser Leu Gly Tyr Cys Met 320325 330 Leu Lys Trp Leu Tyr Gly Phe Leu Pro Trp Thr Asn Cys Leu Pro 335340 345 Asn Thr Glu Asp Ile Met Lys Gln Lys Gln Lys Phe Val Asp Lys 350355 360 Pro Gly Pro Phe Val Gly Pro Cys Gly His Trp Ile Arg Pro Ser 365370 375 Glu Thr Leu Gln Lys Tyr Leu Lys Val Val Met Ala Leu Thr Tyr 380385 390 Glu Glu Lys Pro Pro Tyr Ala Met Leu Arg Asn Asn Leu Glu Ala 395400 405 Leu Leu Gln Asp Leu Arg Val Ser Pro Tyr Asp Pro Ile Gly Leu 410415 420 Pro Met Val Pro 8 99 PRT Homo sapiens 2605059 8 Met Pro Leu GluGlu Val Leu Pro Asp Val Ser Pro Gln Ala Leu 1 5 10 15 Asp Leu Leu GlyGln Phe Leu Leu Tyr Pro Pro His Gln Arg Ile 20 25 30 Ala Ala Ser Lys AlaLeu Leu His Gln Tyr Phe Phe Thr Ala Pro 35 40 45 Leu Pro Ala His Pro SerGlu Leu Pro Ile Pro Gln Arg Leu Gly 50 55 60 Gly Pro Ala Pro Lys Ala HisPro Gly Pro Pro His Ile His Asp 65 70 75 Phe His Val Asp Arg Pro Leu GluGlu Ser Leu Leu Asn Ser Glu 80 85 90 Leu Ile Arg Pro Phe Ile Leu Glu Gly95 9 138 PRT Homo sapiens 3000825 9 Met Trp Val Val Pro Pro Ile Gly AlaGlu Phe Leu Gly Thr Glu 1 5 10 15 Lys Gly Gly Leu Arg Asp Gln Lys ThrPro Asp Asp His Glu Ala 20 25 30 Glu Thr Gly Ile Lys Ser Lys Glu Ala ArgLys Tyr Ile Phe Asn 35 40 45 Cys Leu Asp Ala Cys Val Gln Val Asn Met ThrThr Asp Leu Glu 50 55 60 Gly Ser Asp Met Leu Val Glu Lys Ala Asp Arg ArgGlu Phe Ile 65 70 75 Asp Leu Leu Lys Lys Met Leu Thr Ile Asp Ala Asp LysArg Ile 80 85 90 Thr Pro Ile Glu Thr Leu Asn His Pro Phe Val Thr Met ThrHis 95 100 105 Leu Leu Asp Phe Pro His Ser Thr His Val Lys Ser Cys PheGln 110 115 120 Asn Met Glu Ile Cys Lys Arg Arg Val Asn Met Tyr Asp ThrVal 125 130 135 Asn Gln Ser 10 1427 DNA Homo sapiens 119819 10cggagccaca gtggctccac cccccacctt cacgcactcc cacggtggta atcccgaaag 60gctgggtggc tgggctgacg gtaattcccg gggggggtca agtgccccaa actgctcttg 120gtgaaaggat gctgtcttcc ccgaatggcc acttccgcct gccttagctt gggctgagag 180gggacagaga gcaccctgag gcgggccggc caggtcttcc cactcctaat ggagctgtgg 240ggagtggggc cacaggcggg gaggcaggga gagtagtgag tagctggtgc caaggggcgc 300tggcgccaca ttctggtgtc catgggagcc ctggggcccg gagaggcctc ttccctggcg 360gctgtgcagg gaaacctcca cttcatgctg actggggcgg gcgacaggaa ccctggggtg 420accctggctc tgacagcaga ccggtaagct gtccaaaaac aagaggaaga agatgaggcg 480caaacggaaa cagcagaagc ggctgctgga ggagcggctg cgggacctgc agaggctgga 540ggccatggag gctgccaccc aggctgagga ctctggcttg agactagacg ggggcagcgg 600ctccacatcc tcttcaggct gtcaccccgg gggcgccaga gcaggtccct ccccagcctc 660ttcctccccc gccccagggg gcggccgtag cctcagcgcg ggctcacaga cctcaggctt 720ctccggctcc ctcttctctc ctgcctcctg ctccatcctc tccggctcgt ccaatcagcg 780agagaccggg ggcctcctgt cgcctagcac accattcggt gcctcgaacc tcctggtgaa 840ccccctggag ccccaaaatg cagataagat caagatcaag atcgcagacc tgggcaacgc 900ctgctgggtg cacaagcact tcacggaaga catccagact cggcagtacc gggccgtcga 960ggtgctgatc ggcgccgaat acggcccccc ggcagacatc tggagcacag cctgcatggc 1020cttcgagctg gccactggtg actacctgtt cgagccgcat tctggagaag actacagtcg 1080tgatgaggac cacatcgctc acatagtgga gcttctgggg gacatccccc cagccttcgc 1140cctctcaggc cgctattccc gggagttctt caaccggaga ggagagctgc ggcacatcca 1200caatctcaag cactggggcc tgtacgaggt actcatggaa aagtacgagt ggcccctaga 1260gcaggccaca cagttcagcg cctttctgct gcccatgaat gagtacatcc ccgaaaagcg 1320ggccagtgcc cgtgactgcc tccagcaccc ctggctccaa ccctagggcc cggctgtggc 1380tccacctcca gctctccgtg cctttaaggg aaaagcggga cagctcc 1427 11 1586 DNAHomo sapiens 132750 11 gctcattgac tcttttgtct tctttcctct cgggggtgaggtcagattta ccaccaaaat 60 gcatgcagga gatcccgcaa gagcaaatca aggagatcaagaaggagcag ctttcaggat 120 ccccgtggat tctgctaagg gaaaatgaag tcagcacactttataaagga gaataccaca 180 gagctccagt ggccataaaa gtattcaaaa aactccaggctggcagcatt gcaatagtga 240 ggcagacttt caataaggag atcaaaacca tgaagaaattcgaatctccc aacatcctgc 300 gtatatttgg gatttgcatt gatgaaacag tgactccgcctcaattctcc attgtcatgg 360 agtactgtga actcgggacc ctgagggagc tgttggatagggaaaaagac ctcacacttg 420 gcaagcgcat ggtcctagtc ctgggggcag cccgaggcctataccggcta caccattcag 480 aagcacctga actccacgga aaaatcagaa gctcaaacttcctggtaact caaggctacc 540 aagtgaagct tgcaggattt gagttgagga aaacacagacttccatgagt ttgggaacta 600 cgagagaaaa gacagacaga gtcaaatcta cagcatatctctcacctcag gaactggaag 660 atgtatttta tcaatatgat gtaaagtctg aaatatacagctttggaatc gtcctctggg 720 aaatcgccac tggagatatc ccgtttcaag gctgtaattctgagaagatc cgcaagctgg 780 tggctgtgaa gcggcagcag gagccactgg gtgaagactgcccttcagag ctgcgggaga 840 tcattgatga gtgccgggcc catgatccct ctgtgcggccctctgtggat gaaatcttaa 900 agaaactctc caccttttct aagtagtgta tcaaaatctaaaccaaggag tctctggaca 960 agaagctggg agaggcacaa actggacatc tctctctctcatatccttcg gcattgggtt 1020 atctatggga gcaaggagtg ggcacgcttc tctgttacaaatagaaaacg attccagtca 1080 tacaggacac atcccactcc aaatgatatt tccaaaaacatacctctgac agtaactttg 1140 atagatggtt tgtcaaatgt atctttctgg gtatccacacctcttggcaa tgaaatttgc 1200 agctcctccc ttccataaat gaagtctctt tccccaccatttgaatctgg gctggcactg 1260 tgacttgatt tgatcaatag aatgtggaag aagtgactgtatgccagttc caagcctagg 1320 tttcaagagg ccttataaat gtctgttgga accttacccagccatgaaca tgttgagtga 1380 gcatgctgga gaatgagaga ccacatgaag cagaaacatgctttcctagc tgaagtcata 1440 ctagcccaac caacatggca gctaacacat gaatgaggccaatcaagacc agaagaacca 1500 ctcaagcaga tcccagccca aattgcccat tcacacaatcaggagctaaa taaattactg 1560 ttgtcttaac actaaaaaaa aaaaaa 1586 12 1574 DNAHomo sapiens 507669 12 cgacggcgaa gggagctgag actgtccagg cagccaggttaggccaggag gaccatgtga 60 atggggccag aaggctcccg ggctgggcag ggaccatgggctgtggctgc agctcacacc 120 cggaagatga ctggatggaa aacatcgatg tgtgtgagaactgccattat cccatagtcc 180 cactggatgg caagggcacg ctgctcatcc gaaatggctctgaggtgcgg gacccactgg 240 ttacctacga aggctccaat ccgccggctt ccccactgcaagacaacctg gttatcgctc 300 tgcacagcta tgagccctct cacgacggag atctgggctttgagaagggg gaacagctcc 360 gcatcctgga gcagagcggc gagtggtgga aggcgcagtccctgaccacg ggccaggaag 420 gcttcatccc cttcaatttt gtggccaaag cgaacagcctggagcccgag gccaacctca 480 tgaagcagct gcaacaccag cggctggttc ggctctacgctgtggtcacc caggagccca 540 tctacatcat cactgaatac atggagaatg ggagtctagtggattttctc aagacccctt 600 caggcatcaa gttgaccatc aacaaactcc tggacatggcagcccaaatt gcagaaggca 660 tggcattcat tgaagagcgg aattatattc atcgtgaccttcgggctgcc aacattctgg 720 tgtctgacac cctgagctgc aagattgcag actttggcctagcacgcctc attgaggaca 780 acgagtacac agccagggag ggggccaagt ttcccattaagtggacagcg ccagaagcca 840 ttaactacgg gacattcacc atcaagtcag atgtgtggtcttttgggatc ctgctgacgg 900 aaattgtcac ccacggccgc atcccttacc cagggatgaccaacccggag gtgattcaga 960 acctggagcg aggctaccgc atggtgcgcc ctgacaactgtccagaggag ctgtaccaac 1020 tcatgaggct gtgctggaag gagcgcccag aggaccggcccacctttgac tacctgcgca 1080 gtgtgctgga ggacttcttc acggccacag agggccagtaccagcctcag ccttgagagg 1140 ccttgagagg ccctggggtt ctcccccttt ctctccagcctgacttgggg agatggagtt 1200 cttgtgccat agtcacatgg cctatgcaca tatggactctgcacatgaat cccacccaca 1260 tgtgacacat atgcaccttg tgtctgtaca cgtgtcctgtagttgcgtgg actctgcaca 1320 tgtcttgtac atgtgtagcc tgtgcatgta tgtcttggacactgtacaag gtaccccttt 1380 ctggctctcc catttcctga gaccacagag agaggggagaagcctgggat tgacagaagc 1440 ttctgcccac ctacttttct ttcctcagat catccagaagttcctcaagg gccaggactt 1500 tatctaatac ctctgtgtgc tcctccttgg tgcctggcctggcacacatc aggagttcaa 1560 taaatgtctg ttga 1574 13 1866 DNA Homo sapiens1439938 13 cgggaggaag agggagaggg agaccgggac gagaccgggg ctgtggtgcggagagaggct 60 gagacggaga agaggagagg cagagagggc gcggggaccg tcagcagcaccttagctaca 120 atcgttcagc tattctcgga agagagaagg gagagggagg aggccggggcgggagtgggg 180 gctgtcaccc tcggaccccg gcgtgagagg ggccgtgcgg ccggacgtcctcggggtggg 240 cccccagtcg gtggccgaag acctacagct caggcccctg ggtcccaaatttccaggctt 300 tgcccctcct cctttctcag atacccgggt aacagtcctc atagtccagatatccgggac 360 tcgggtccca acctctctaa acctgggtct ctgtttcata gaatttcaaatatcaggttc 420 aggcccctgc gtgcaccagt atccggggtt cattccccgg gcgttcagatatcggattca 480 gtctccatcc cgttcagata ttcggggttc agaccccaca atcagaaatccggaattcgg 540 cagctgtcgc cctcgacgag ggggaggact ggaccgcgag gtcagattaggttgtcaccc 600 cctcccctcc aggggaggct tcccgggccc gcccctcagg aagggcgaaagccgaggaag 660 aggtggcaag gggaaaggtc tccttgcccc tctccctgct tggcagagccgctggaggac 720 cccaggcgga agcggaggcg ctggggcacc atagtgaccc ctaccaggccaggccccact 780 ctcagggccc ccaggggcca ccatgccagc tgggggccgg gccgggagcctgaaggaccc 840 agatgtggct gagctcttct tcaaggatga cccagaaaag ctcttctctgacctccggga 900 aattggccat ggcagctttg gagccgtata ctttgcccgg gatgtccggaatagtgaggt 960 ggtggccatc aagaagatgt cctacagtgg gaagcagtcc aatgagaaatggcaagacat 1020 catcaaggag gtgcggagac gaaggagagt agggagggag gatgaagagagataaggggg 1080 agaaaagaga ggggcatgag agtggagcgg agctaagaag gggtagaagagagagtgggt 1140 gaaggggaag agacgtagag aaagtgtgga gagaggaaag gcatagcgagagaacgaggg 1200 agagagaagt ggaaggggga agtaagagag gataagagga acgagaggaggggaagggtg 1260 gggacgagaa cgaagagcat gatggagagg aaagatagag aagagaggaagtggaggcag 1320 ttagggggca tggaggagag agagagatga gggagagtgg gagcacggggcggatggacg 1380 gggtggagaa gaagagaggg aggagatgag aggaggaaga ggtgggagaaccgagcgagg 1440 gaaaagatgg aggaggcagt agagagggtg tgcaaggggt gaaaagaaagaagaaggaaa 1500 aggatggagg gagtgaaggt aggagacgag gaggagggat gggagagaatggagggtagc 1560 gtgtggatgg tgagtggtag agaatagtga gatggtgaga agcggagaaaggcagcagag 1620 gatgggggtg aagcgggaag caaagacaat aggggatgga ggaggagaggagcaggagga 1680 agacgaagag cgaagggctt gaaagaggga gaagagagta gtaaggggtaggtatgtaga 1740 tgcgagtagg agaggaagag aaggaatgaa tgagagagag tagagagtagagagagaacg 1800 aaggaacggg gcagagggag aggaaggaca gaaggagaag agaacaatcgaagaatgaga 1860 gtgttt 1866 14 1498 DNA Homo sapiens unsure 1350, 1355,1372, 1444 a or g or c or t, unknown, or other 14 ctcccctccc agcaaccggtctggcggcgg cgcggcagta aaactgagga ggcggagcaa 60 gacggtcggg gctgcttgctaactccagga acaggtttaa gtttttgaaa ctgaagtagg 120 tctacacagt aggaactcatgtcatttctt gtaagtaaac ccagagcgaa tccaggacca 180 atgatgcgag ctcagagtcaatagcatcct tctctaaaca ggaggtcatg agtagctttc 240 tgccagaggg agggtgttacgagctgctca ctgtgatagg caaaggattt gaggacctga 300 tgactgtgaa tctagcaaggtacaaaccaa caggagagta cgtgactgta cggaggatta 360 acctagaagc ttgttccaatgagatggtaa cattcttgca gggcgagctg catgtctcca 420 aactcttcaa ccatcccaatatcgtgccat atcgagccac ttttattgca gacaatgagc 480 tgtgggttgt cacatcattcatggcatacg gttctgcaaa agatctcatc tgtacacact 540 tcatggatgg catgaatgagctggcgattg cttacatcct gcagggggtg ctgaaggccc 600 tcgactacat ccaccacatgggatatgtac acaggagtgt caaagccagc cacatcctga 660 tctctgtgga tgggaaggtctacctgtctg gtttgcgcac aacgctcagc atgataagcc 720 atgggcagcg gcagcgagtggtccacgatt ttcccaagta cagtgtcaag gttctgccgt 780 ggctcagccc cgaggtcctccagcagaatc tccagggtta tgatgccaag tctgacatct 840 acagtgtggg aatcacagcctgtgaactgg ccaacggcca tgtccccttt aaggatatgc 900 ctgccaccca gatgctgctagagaaactga acggcacagt gccctgcctg ttggatacca 960 gcaccatccc cgctgaggagctgaccatga gcccttcgcg ctcagtggcc aactctggcc 1020 tgagtgacag cctgaccaccagcacccccc ggccctccaa cggtgactcg ccctcccacc 1080 cctaccaccg aaccttctccccccacttcc accactttgt ggagcagtgc cttcagcgca 1140 acccggatgc caggtatccctgctggcctg ggcctgggct tcgggagagc agagggtgct 1200 caggagggta aggccagggtgtgaagggac ttacctccca aaggttctgc aggggaatct 1260 ggagctacac acaggagggatcagctcctg ggtgtgtcag aggccagcct ggggagctct 1320 ggccactgct tcccatgagctgagggagan ggagnaggga cccgaggctg angcataagt 1380 ggcaggattt tcggaagctggggacacggc agtgatgctg cggtctctcc ctcccttacc 1440 tcangctcag tgcagcaccctctgaacact ctttctcagc agtatcgtag ccttcgtt 1498 15 1846 DNA Homo sapiens1567782 15 taggaattcg tcgacccacg cgatccgccg tcagaagact gccacacctagactgatgct 60 tattagtcat caccgttatt cctactaacg tcctgtgtca ctgagttttttaaatgtcta 120 gcatatctgt aaagatgcct tagaaaaaga atcatggaga agtatgttagactacagaag 180 attggagaag gttcatttgg aaaagccatt cttgttaaat ctacagaagatggcagacag 240 tatgttatca aggaaattaa catctcaaga atgtccagta aagaaagagaagaatcaagg 300 agagaagttg cagtattggc aaacatgaag catccaaata ttgtccagtatagagaatca 360 tttgaaggaa ttttggactg gtttgtacag atatgtttgg ccctgaaacatgtacatgat 420 agaaaaattc ttcatcgaga cattaaatct cagaacatat ttttaactaaagatggaaca 480 gtacaacttg gagattttgg aattgctaga gttcttaata gtactgtagagctggctcga 540 acttgcatag ggaccccata ctacttgtca cctgaaatct gtgaaaacaaaccttacaat 600 aataaaagtg acatttgggc tctggggtgt gtcctttatg agctgtgtacacttaaacat 660 gcttttgaag ctggcagtat gaaaaacctg gtactgaaga taatatctggatcttttcca 720 cctgtgtctt tgcattattc ctatgatctc cgcagtttgg tgtctcagttatttaaaaga 780 aatcctaggg atagaccatc agtcaactcc atattggaga aaggttttatagccaaacgc 840 attgaaaagt ttctctctcc tcagcttatt gcagaagaat tttgtctaaaaacattttcg 900 aagtttggat cacagcctat accagctaaa agaccagctt caggacaaaactcgatttct 960 gttatgcctg ctcagaaaat tacaaagcct gccgctaaat atggaatacctttagcatat 1020 aagaaatatg gagataaaaa attacacgaa aagaaaccac tgcaaaaacataaacaggcc 1080 catcaaactc cagagaagag agtgaatact ggagaagaaa ggaggaaaatatctgaggaa 1140 gcagcaagaa agagaaggct ggaatttatt gaaaaagata aggaacggtaggatcagatt 1200 attagtttaa tgaaggctga acaaatgaaa aggcaagaca aggaaaggttggaaagaata 1260 aatagggcca gggaacaagg atggagaaat gtgctaagtg ctggtggaagtggtgaagta 1320 aaggtaggca ttttatacca atatggttat actaccattt tcccctccagttccaccttg 1380 ttctataaaa tgcatgtact tgggattttc tttctttctt tagtgtacaattaattttta 1440 cctagaattc tttaacattt attatgaata cttagctttc ctgcatgtatctgatatgta 1500 acttgtgttg ctgttatgtg actatactca aaattgcttt aaaagttttttgtgaagact 1560 atgataacat tattcctgtc aggaattttt aaaaattatg tacaattcatgacactgcag 1620 cctaaaatcg ttctgtaatt tcatgtagcc ttgaagatta agttctcagaagatgcttct 1680 taaatccgat ccctgttgtc tctccaattt catcaccatt cattcccctaccacatactg 1740 ggaagggcct attccatggc ggaaatgaag ggccataatt tgtaggttttccattaccaa 1800 taatgggggg ttggcccaaa tcctactttt gggcctttgg aacctt 184616 1721 DNA Homo sapiens 2295842 16 agttggacga ggctcagtga aagttttcgctgggcaactg agaaggtcgc tgtcaagatg 60 gagtttccaa cccagtaaat ccaagggccagaccgtgacc tcataaagca tgatctcctt 120 ctgtccagac tgtggcaaaa gtatccaagcggcattcaaa ttctgcccct actgtggaaa 180 ttctttgcct gtagaggagc atgtagggtcccagaccttt gtcaatccac atgtgtcatc 240 cttccaaggc tccgggagca gacccccaacccccaaaagc agccctcaga agaccaggaa 300 gagccctcag gtgaccaggg gtagccctcagaagaccagc tgtagccctc agaagaccag 360 gcagagccct cagacgctga agcggagccgagtgaccacc tcacttgaag ctttgcccac 420 agggacagtg ctgacagaca agagtgggcgacagtggaag ctgaagtcct tccagaccag 480 ggacaaccag ggcattctct atgaagctgcacccacctcc accctcacct gtgactcagg 540 accacagaag caaaagttct cactcaaactggatgccaag gatgggcgct tgttcaatga 600 gcagaacttc ttccagcggg ccgccaagcctctgcaagtc aacaagtgga agaagctgta 660 ctcgacccca ctgctggcca tccctacctgcatgggtttc ggtgttcacc aggacaaata 720 caggttcttg gtgttaccca gcctggggaggagccttcag tcggccctgg atgtcagccc 780 aaagcatgtg ctgtcagaga ggtctgtgctgcaggtggcc tgccggctgc tggatgccct 840 ggagttcctc catgagaatg agtatgttcatggaaatgtg acagctgaaa atatctttgt 900 ggatccagag gaccagagtc aggtgactttggcaggctat ggcttcgcct tccgctattg 960 cccaagtggc aaacacgtgg cctacgtggaaggcagcagg agccctcacg agggggacct 1020 tgagttcatt agcatggacc tgcacaagggatgcgggccc tcccgccgca gcgacctcca 1080 gagcctgggc tactgcatgc tgaagtggctctacgggttt ctgccatgga caaattgcct 1140 tcccaacact gaggacatca tgaagcaaaaacagaagttt gttgataagc cggggccctt 1200 cgtgggaccc tgcggtcact ggatcaggccctcagagacc ctgcagaagt acctgaaggt 1260 ggtgatggcc ctcacgtatg aggagaagccgccctacgcc atgctgagga acaacctaga 1320 agctttgctg caggatctgc gtgtgtctccatatgacccc attggcctcc cgatggtgcc 1380 ctaggtggaa tccagaactt tccatttgcagtgtgcaaca gaaaaaaaaa aatgaagtaa 1440 tgtgactcaa ggcctgctgt ttaatcacagataagcttct agaacaagcc ctggaatgtg 1500 cattcctgcc actggtttca ggatactcatcagtcctgat tagcctcccg gagggcccca 1560 gtttccctcc cgtgaatgtg aagttccccatcttggtggc ctgcccttca gccagtgtcc 1620 tagcaaagct ggatggggtt gggccggcccacagggggga cccctcctac ccttgacacc 1680 tctgtgcttt ggtaataaat tgttttaccagaaaaaaaaa a 1721 17 1985 DNA Homo sapiens 2605059 17 ttcgcatcttgggcacccca aaccctcaag tctggccggt ttgtaggggc ccttggtgag 60 gtgggtgtggggcaggttta ctccactccc aacagcaagt aaccactccc tcccctgaac 120 cttctctctcctggccccaa ccccccttga tggacaggga ccactgtcct ggcccaactc 180 agggcttcctccttcctgct gtcatttggg ttggggtaga tcctgtcctt tgtccctttt 240 caccctagtacacacatgtg cagtgtctca gcaagctgtg cacagagtcg tcatctgaga 300 gggcaaggggatggatgaag gaatacaggg gtgggtgagt gaatgaatga tgggtcaggg 360 agacacatggatgggagagc accccccatg tgagtgtgtg ttaggggctg agagttgaca 420 gcagagagcatggcaagggt cgggaactac tctcattgta ccctgttcct tctccctggc 480 ccaggagctcactgagctgc cggactacaa caagatctcc tttaaggagc aggtgcccat 540 gcccctggaggaggtgctgc ctgacgtctc tccccaggca ttggatctgc tgggtcaatt 600 ccttctctaccctcctcacc agcgcatcgc agcttccaag gctctcctcc atcagtactt 660 cttcacagctcccctgcctg cccatccatc tgagctgccg attcctcagc gtctaggggg 720 acctgcccccaaggcccatc cagggccccc ccacatccat gacttccacg tggaccggcc 780 tcttgaggagtcgctgttga actcagagct gattcggccc ttcatcctgg aggggtgaga 840 agttggccctggtcccgtct gcctgctcct caggaccact cagtccacct gttcctctgc 900 cacctgcctggcttcaccct ccaaggcctc cccatggcca cagtgggccc acaccacacc 960 ctgccccttagcccttgcga gggttggtct cgaggcagag gtcatgttcc cagccaagag 1020 tatgagaacatccagtcgag cagaggagat tcatggcctg tgctcggtga gccttacctt 1080 ctgtgtgctactgacgtacc catcaggaca gtgagctctg ctgccagtca aggcctgcat 1140 atgcagaatgacgatgcctg ccttggtgct gcttccccga gtgctgcctc ctggtcaagg 1200 agaagtgcagagagtaaggt gtccttatgt tggaaactca agtggaagga agatttggtt 1260 tggttttattctcagagcca ttaaacacta gttcagtatg tgagatatag attctaaaaa 1320 cctcaggtggctctgcctta tgtctgttcc tccttcattt ctctcaaggg aaatggctaa 1380 ggtggcattgtctcatggct ctcgtttttg gggtcatggg gagggtagca ccaggcatag 1440 ccacttttgccctgagggac tcctgtgtgc ttcacatcac tgagcactca tttagaagtg 1500 agggagacagaagtctaggc ccagggatgg ctccagttgg ggatccagca ggagaccctc 1560 tgcacatgaggctggtttac caacatctac tccctcagga tgagcgtgag ccagaagcag 1620 ctgtgtatttaaggaaacaa gcgttcctgg aattaattta taaatttaat aaatcccaat 1680 ataatcccagctagtgcttt ttccttatta taatttgata aggtgattat aaaagataca 1740 tggaaggaagtggaaccaga tgcagaagag gaaatgatgg aaggacttat ggtatcagat 1800 accaatatttaaaagtttgt ataataataa agagtatgat tgtggttcaa ggataaaaac 1860 agactagagaaacttattct tagccatcct ttatttttat tttatttatt ttttgatgga 1920 gtcttgctctgttgcccact gcaattcaag ccttggtgac agactctggt ctcaaaaaaa 1980 aaaaa 198518 661 DNA Homo sapiens 3000825 18 tgaggagtga tgaaagctgc atttcaacttaactgatgaa agcaggagca gtttacatcc 60 tgtcattcag atatatttgc aggtcccagcagcagccctc tccccttcct ggggcacagc 120 ccctctctgc ctttcctgca gagagaaaagccacatcctg tgggcaatga caacatgtgg 180 gtggtgcctc ccataggggc agagttcctgggaactgaga aagggggctt gagagatcag 240 aagacaccag atgaccatga agcagagacagggattaagt caaaagaagc aagaaagtac 300 attttcaact gtttagatgc ttgcgtccaggtgaacatga cgacagattt ggaagggagc 360 gacatgttgg tagaaaaggc tgaccggcgggagttcattg acctgttgaa gaagatgctg 420 accattgatg ctgacaagag aatcactccaatcgaaaccc tgaaccatcc ctttgtcacc 480 atgacacact tactcgattt tccccacagcacacacgtca aatcatgttt ccagaacatg 540 gagatctgca agcgtcgggt gaatatgtatgacacggtga accagagcta aacctagccc 600 caaacccctc tgccgaatat cctcgctcgagggccaaatt ccctatagtg gtcgtattac 660 g 661

What is claimed is:
 1. A purified polypeptide comprising an amino acidsequence selected from the group consisting of: a) an amino acidsequence selected from the group consisting of SEQ ID NO:1-9, b) anaturally-occurring amino acid sequence having at least 95% sequenceidentity to the sequence selected from the group consisting of SEQ IDNO:1-2, 4, 6-7 and 9, wherein said amino acid sequence encodes apolypeptide having protein kinase activity, c) a biologically-activefragment of the amino acid sequence selected from the group consistingof SEQ ID NO:1-2, 4, 6-7 and 9, wherein said biologically-activefragment encodes a polypeptide having protein kinase activity, and d) animmunogenic fragment of the amino acid sequence selected from the groupconsisting of SEQ ID NO:1-2, 4, 6-7 and 9, wherein said polypeptides iscapable of generating antibody that specifically binds to thepolypeptide selected from the group consisting of SEQ ID NO:1-2, 4, 6-7and
 9. 2. An isolated polypeptide of claim 1, having a sequence selectedfrom the group consisting of SEQ ID NO:1-9.
 3. A composition comprisingan effective amount of a polypeptide of claim 1 and a pharmaceuticallyacceptable carrier.
 4. A composition of claim 3, wherein the polypeptidehas the sequence selected from the group consisting of SEQ ID NO: 1-9.